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Olga V. Kovalchukova BASIC BIOINORGANIC CHEMISTRY Educational and methodical complex Moscow Peoples’ Friendship University of Russia 2013 УДК ББК Б Утверждено РИС Ученого совета Российского университета дружбы народов Издание подготовлено в рамках реализации Программы стратегического развития РУДН на 2012–2016 гг. Б Ковальчукова, О. В. Основы бионеорганической химии (Basic Bioinorganic Chemistry) [Текст] : учебно-методический комплекс / О. В. Ковальчукова. – М.: РУДН, 2013. – 214 с. Представленный учебно-методический комплекс обеспечивает реализацию магистерской программы «Химия окружающей среды» и включает краткое описание курса, программу, систему оценки знаний, краткий конспект лекций, описание основных лабораторных работ и материалы и вопросы для подготовки к текущему и итоговому контролю знаний. The presented educational and methodical complex provides the realization of the Master’s Program “Chemistry of the Environment”. It includes the short description of the course, its syllabus, grading procedure, lecture notes, description of the major lab. experiments, as well as the materials and questions for the mid-semester and final controls. ISBN 978-5-209- © Ковальчукова О.В., 2013 © Российский университет дружбы народов, Издательство, 2013 2 INTRODUCTION The course of the Basic Bioinorganic Chemistry is at the gate-way of inorganic chemistry and biochemistry. It describes the mutual relationship between these two sub-disciplines, with focus upon the function of inorganic “substances“ in living systems, including the transport, speciation and, eventually, mineralisation of inorganic materials, and including the use of inorganics in medicinal therapy and diagnosis. These “substances” can be metal ions (such as Na+, Fe2+, Fe3+), composite ions (e.g. MoO42-), coordination compounds (like hemoglobin and cobalamine), or inorganic molecules (CO, NO, O3). The course begins with the principles of coordination chemistry and a survey of the abilities of various functional groups within proteins and nucleic acids to form coordination complexes with metal ions. The reactivity of coordination compounds of metal ions is discussed in the context of the reaction mechanisms of specific metalloenzymes. A part of the course is devoted to medicallyrelevant topics (e.g. the mechanisms by which organisms obtain required metal ions from their environment, the toxicity of metals, and use of metal containing compounds in treating cancer and other deceases). 3 4 Content Basic Bioinorganic Chemistry (Study Guide)……………………………….. 1. 2. 3. 4. 4.1. 4.2. 4.3. 5. 6. 7. 8. 9. 10. Aims and Goals……………………………………… Requirements to results of development of the discipline………………………………………………. Filling of the discipline and study kinds…………… The discipline maintenance…………………………. The maintenance of sections of the discipline………. The sections of the discipline and interdisciplinary connections with the other disciplines………………. The discipline maintenance and kinds of employment……………………………………………. Laboratory practical work…………………………… Subjects of research presentations…………………… Discipline information support……………………… Discipline Material support………………………….. Methodical recommendations about the organization of studying of the discipline………………………….. Grading……………………………………………….. 7 9 9 10 11 11 12 13 13 15 15 16 16 17 Supplementary Materials……………………. 19 Glossary of Terms……………………………. 21 Lecture notes………………………………….. 63 Chapter 1 Introduction to Bioinorganic Chemistry…….. Chapter 2 Transition Metal Chemistry…………………. 65 88 5 Chapter 3 Structure, properties, and functions of metal complexes of proteins and nucleic acids……... Chapter 4 Transition Metal Storage, Transport and Biomineralization……………………………… Chapter 5 Biological dioxygen transport system………... Chapter 6 Bioinorganic Chemistry of dinitrogen fixation………………………………………….. 107 126 138 161 Experiments in Bioinorganic Chemistry……. 169 6 Instructions for Research topic: Presentation, Questions and Term Paper… 209 Exam Questions…………………………….. 211 List of citations…………………………. 213 7 BASIC BIOINORGANIC CHEMISTRY Study Guide 8 1. Aims and Goals The aims and goals of the discipline are to provide students in basic knowledges in properties of bio-essential inorganic elements and their behavior in biological systems The discipline is included in the variational part of the Professional cycle. The subject maintenance is based on such disciplines as “General Chemistry”, “Inorganic Chemistry”, “Organic Chemistry”, “Analytical Chemistry”, and “Physical Chemistry”. 2. Requirements to results of development of the discipline The teaching process is faced on formation of the following competences: ОК-1-6, ПК-1-7 As a result of studying of the discipline, students must: Know: theoretical bases of bioinorganic chemistry, composition and structure of complexes of metals with constituents of biologically active molecules, principles of action of metalloenzymes Be able to: predict the electronic and spatial structures of active sites of metalloenzymes; determine the composition and stability of metallic complexes with the organic ligands – models of biologically active molecules. Own: the methods of investigation of biologically active metallic complexes. 9 3. Filling of the discipline and study kinds The total labor input includes 4 credits. The study kinds Auditoriums (in total) Including: Lectures Practicals Tutorials Laboratory Out-class work (in total) Including: Academic year project Calculations Research presentation Slide presentation Other types of work (including interactive) Final examination Hours 72 I 72 36 36 36 72 36 72 8 20 20 20 4 The total labor input: Hours Credits 10 144 4 Semester II III IV 4. The discipline maintenance 4.1. The maintenance of sections of the discipline No 1. The section Introduction. 2. Transitional metal chemistry 3. Structure, properties and functions of metal complexes of proteins and nucleic acids. 4. Transition-Metal Storage, Transport, and Biomineralization 5. Biological dioxygen The maintenance Historical background and current relevance. Bio-essential elements. Fundamentals of Coordination Chemistry. Crystal Field Theory: Ligands & Geometries: Oh; Td; Sq.Pl. 10Dq; spectrochemical series; Template effect; Chelate effect; Hard/Soft Acids/Bases Model Compounds Composition of proteins and nucleic acids. Aminoacids. Nucleic bases. Electron donating groups and features of their complex formation. Chelating properties of aminoacids. Complexes of transition metals with proteins and nucleic acids and their biological applications. Biological significance of metals. Chemical Properties Relative to Storage and Transport. Role of ferritin and transferring in storage and transport of iron. Iron biomineralization. Vitamins and Coferments. Electronic structure and oxidative 11 transport system 6. Bioinorganic Chemistry of dinitrogen fixation. properties if dioxygen. Requirements for Effective Oxygen Carriers. Free-Radical Autoxidation. Role of enzymes. Dioxygen toxicity. Defense and Repair Systems. Electron transfers in biology. Nitrogenase, Мо-Fе- and Feprotein. Dinitrogen complexes with transitional metals. Chlorophyll as Mg-comples, photosynthesis. 4.2. The sections of the discipline and interdisciplinary connections with the other disciplines No Name of the connecting discipline 1. Chemistry of the enviroments and ecological problems Instrumental methods of analysis of natural compounds Physical and chemical methods of studying of chemical compounds Chemical toxicology Philosophical problems of chemistry 2. 3. 4. 5. 12 No of the section if the studying discipline 1 2 3 4 5 6 + + + + + + + + 4.3. The discipline maintenance and kinds of employment No 1. 2. 3. 4. 5. 6. The name of the section of the discipline Introduction. Transitional metal chemistry Structure, properties and functions of metal complexes of proteins and nucleic acids. Transition-Metal Storage, Transport, and Biomineralization Biological dioxygen transport system Bioinorganic Chemistry of dinitrogen fixation. OutLect Pract Lab Tut class work 6 6 6 4 6 In total, hours 12 16 6 10 26 6 8 10 24 6 4 6 16 6 4 4 14 10 5. Laboratory practical work No No of the Name of the laboratory practical section of the work discipline 1. 1 Electronic formulae of atoms and ions. Molecular diagrams of labor input (hours) 2 13 2. 1 3. 2 4. 3 5. 3 6. 4 7. 3 8. 4 9. 5, 6 10. 1–6 14 molecules and molecular ions. Bond orders and multiplicities of electronic states. Electronic and spatial structures and chemical properties of coordinate compounds. Redox-properties of complexes of transition metals. Quantum-chemical modeling of tautomeric forms of nucleic acids and their complexes with metals. Determination of ionization constants of amino acids by potentiometry. Determination of stability constants of complexes of transitional metals by potentiometry. Determination of protonation constants of nucleic bases by UVspectrophotometry. Determination of stability constants of complexes of transitional metals with the nuclear bases by UV-spectrophotometry. Determination of coordination sites of O- and N-donating ligands by IR-spectroscopy. Conference on research topics. 2 2 4 4 4 4 4 6 4 6. Subjects of research presentations 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. X-Ray and DNA demonstration. Storage, Transport, and Biomineralization of Zink. Storage, Transport, and Biomineralization of Copper. Storage, Transport, and Biomineralization of Cobalt. Molybdenum enzymes. Selective organic synthesis catalyzed by organometallic compounds. Mechanisms of metallic toxicity. Chelation therapy and metallotherapy. Manganese complexes as Catalysts for Peroxynitrite Decomposition. Oxidative stress and its influence on organisms. Metals and Health. 7. Discipline information support Required Textbook: Ivano Bertini, Harry B. Gray,, Bioinorganic Chemistry, Mill Valley, California, 1994. (available at the Department of General Chemistry) Supplementary readings: i) S.J. Lippard, J.M. Berg, Principles of Bioinorganic Chemistry, U.S.B., 1994. ii) Frausto da Silva, J. J. R. and Williams, R. J. P. The Biological Chemistry of the Elements: the Inorganic Chemistry of Life (2nd. edition, Oxford, 2001). iii) Roat-Malone, R. M. Bioinorganic Chemistry: A Short Course (Wiley, 2002). 15 iv) Bertini, I.; Gray, H. B.; Stiefel, E. I.; Valentine, J. S., editors. Biological Inorganic Chemistry (University Science Books, 2007). v) Journal of Inorganic Biochemistry (original papers). Computational support: AM1, PM3 Databases, directory and search engines: http://www.sciencedirect.com/science/journal/00063061 http://www.chm.bris.ac.uk/pt/harvey/bioinorganic.htm http://wwwchem.uwimona.edu.jm:1104/courses/CFT.html http://www.pnas.org/content/100/7/3601.full http://books.google.ru/books?id=zGJtXzPINAUC&printsec=f rontcover&hl=ru#v=onepage&q&f=false http://www.adichemistry.com/inorganic/bioinorganic/bioinor ganic-chemistry.html http://simple.wikipedia.org/wiki/ 8. Discipline Material support Chemical equipment and reagents, spectrophotometers, pH-meter, computers IR and UV 9. Methodical recommendations about the organization of studying of the discipline During studying the discipline the students attend lectures, perform lab experiments and prepare a selected research topic for 16 presentation and discussion in the class or in the interactive form. Each kind of study is graded by special No of points. 10. Grading The study kind 1. Lecture attendance 2. Laboratory experiments 3. Research topic In total No of points 95 - 100 86 - 94 69 - 85 61 - 68 51 - 60 31 - 50 0 - 30 No of kinds 18 10 1 Grade 5+ 5 4 3+ 3 2+ 2 No of points 0-2 0-5 0-14 Maximal points 36 50 14 100 ECTS grade A B C D E FX F 17 18 SUPPLEMENTARY MATERIALS 19 20 GLOSSARY OF TERMS1 1 Cited from Glossary of terms used in Bioinorganic Chemistry. Ed. M. W. G. de Bolster. Pure &App. Chem., Vol. 69, No. 6, pp. 1251-1303, 1997. 21 22 Acid See Brensted acid, Lewis acid, hard acid, soft acid. Acidity constant The equilibrium constant for splitting off a hydron from a Brensted acid. Active center The location in an enzyme where the specific reaction takes place. Active site See active center. Aerobe An organism that needs dioxygen for respiration and thus for growth. Albumin A type of protein, especially a protein of blood plasma which transports various substances, including metal ions, drugs and xenobiotics. Ambidentate Ligands, such as (NCS)-, that can bind to a central atom through either of two or more donor atoms are termed ambidentate. Amino-acid residue (in a polypeptide) When two or more amino acids combine to form a peptide, the elements of water are removed, and what remains of each amino acid is called an amino-acid residue. Aminoacid residues are therefore structures that lack a hydrogen atom of the amino group (-NH-CHR-COOH), or the hydroxy moiety of the carboxy group (NH,-CHR-CO-), or both (-NH-CHR-CO-); all units of a 23 peptide chain are therefore amino-acid residues. (Residues of amino acids that contain two amino groups or two carboxy groups may be joined by isopeptide bonds, and so may not have the formulas shown). The residue in a peptide that has an amino group that is free, or at least not acylated by another amino-acid residue (it may, for example, be acylated or formylated), is called N-terminal; it is the N-terminus. The residue that has a free carboxy group, or at least does not acylate another amino-acid residue, (it may, for example, acylate ammonia to give -NHCHR-CO-NH,), is called C-terminal. Anabolism The processes of metabolism that result in the synthesis of cellular components from precursors of low molecular weight. Anaerobe An organism that does not need dioxygen for growth. Many anaerobes are even sensitive to dioxygen. Obligate (strict) anaerobes grow only in the absence of dioxygen. Facultative anaerobes can grow either in the presence or in the absence of dioxygen. Anation Replacement of the ligand water by an anion in a coordination entity. Anti In the representation of stereochemical relationships “anti” means “on opposite 24 sides” of a reference plane, in contrast to “syn” which means “on the same side”. Aquation The incorporation of one or more integral molecules of water into another chemical species with or without displacement of one or more other atoms or groups. See also hydration. ATP adenosine 5’-triphosphate. Auranofin See gold drugs. Azurin An electron transfer protein containing a type 1 copper site, isolated from certain bacteria. Base See Bronsted base, Lewis base, hard base, soft base. Base pairing The specific association between two complementary strands of nucleic acids that results from theformation of hydrogen bonds between the base components of the nucleotides of each strand: A=T and G=C in DNA, A=U and Gr-C (and in some cases Gr-U) in RlVA (the lines indicate the number of hydrogen bonds). Single-stranded nucleic acid molecules can adopt a partially double-stranded structure throughintrastrand base pairing. See also nucleosides. Basicity constant See acidity constant. 25 Beta() sheet Preferentially called a beta pleated sheet; a regular structure in an extended polypeptide chain, stabilized inthe form of a sheet by hydrogen bonds between CO and NH groups of adjacent (parallel or antiparallel)chains. Beta() strand Element of a beta sheet. One of the strands that is hydrogen bonded to a parallel or antiparallel strand to form a beta sheet. Beta() turn A hairpin structure in a polypeptide chain reversing its direction by forming a hydrogen bond between the CO group of amino-acid residue n with the NH group of residue (n+3). Bifunctional ligand A ligand that is capable of simultaneous use of two of its donor atoms to bind to one or more central atoms (see also ambidentate). Binding constant See stability constant. Binding site A specific region (or atom) in a molecular entity that is capable of entering into a stabilizing interaction with another molecular entity. An example of such an interaction is that of an active site in an enzyme with its substrate. Typical forms of interaction are by hydrogen bonding, coordination, and ion pair formation. Two binding sites in different molecular entities are said to be complementary if their 26 interaction is stabilizing. Binuclear Less-frequently used term for the IUPAC recommended: dinuclear. See nuclearit.y Biocatalyst A catalyst of biological origin, typically an enzyme. Bioleaching Extraction of metals from ores or soil by biological processes, mostly by microorganisms. Biomembrane Organized sheet-like assemblies consisting mainly of proteins and lipids (bilayers), acting as highly selective permeability barriers, containing specific molecular pumps and gates, receptors and enzymes. Biomimetic Refers to a laboratory procedure designed to imitate a natural chemical process. Also refers to a compound that mimics a biological material in its structure or function. Biomineralization The synthesis of inorganic crystalline or amorphous mineral-like materials by living organisms. Among the minerals synthesized biologically in various forms of life are: fluoroapatite, hydroxyapatite (Ca5(PO4)3(F, OH)), magnetite (Fe3O4) and calcium carbonate (CaCO3). Biopolymers Macromolecules (including proteins, nucleic 27 acids and polysaccharides) formed by living organisms. Biotransformatio n A chemical transformation mediated by living organisms or enzyme preparations. See also bioconversion. Blue copper protein An electron transfer protein containing a type 1 copper site. Characterized by a strong absorption in the visible region, and an EPR signal with an unusually small hyperfine coupling to the copper nucleus. Bridging ligand A bridging ligand binds to two or more central atoms, usually metals, thereby linking them together to produce polynuclear coordination entities . Bridging is indicated by the Greek letter p appearing before the ligand name and separated by a hyphen. For an example, see FeMocofactor. Brønsted acid A molecular entity capable of donating a hydron to a base (i.e., a “hydron donor”) or the corresponding chemical species. Brønsted base A molecular entity capable of accepting a hydron from an acid (i.e., a “hydron acceptor”) or the corresponding chemical species. Carbon monoxide Enzymes that catalyze the oxidation of carbon monoxide to carbon dioxide. They dehydrogenases 28 contain iron-sulfur clusters and either nickel and zinc, or molybdopterin. Some nickel-containing enzymes are also involved in the synthesis of acetyl coenzyme A from CO2 and H2. Catalase A hemeprotein, which catalyzes the disproportionation of dihydrogen peroxide to O2 and water ; it also catalyzes the oxidation of other compounds, such as ethanol, by dihydrogen peroxide. A nonheme protein containing a dinuclear manganese cluster with catalase activity is often called pseudocatalase. Central atom The atom in a coordination entity that binds other atoms or group of atoms (ligands) to itself, thereby occupying a central position in the coordination entity. Charge-transfer complex An aggregate of two or more molecules in which charge is transferred from a donor to an acceptor. Chelation Chelation involves coordination of more than one sigma-electron pair donor group from the same ligand to the same central atom. The number of coordinating groups in a single chelating ligand is indicated by the adjectives didentate, tridentate, tetradentate, etc. Chlorophyll Part of the photosynthetic systems in green 29 plants. Generally speaking, it can be considered as a magnesium complex of aporphyrin in which a double bond in one of the pyrrole rings (17-18) has been reduced. A fused cyclopentanone ring is also present (positions 13-14-15). In the case of chlorophyll a, the substituted porphyrin ligand further contains four methyl groups in positions 2,7, 12 and 18, a vinyl group in position 3, an ethyl group in position 8 and a -(CH,),CO,R group (R = phytyl, (2-(7R, 11R)-3,7,11,15-tetramethylhexadec-2-en-1yli)n position 17. In chlorophyll b, the group in position 7 is a -CHO group. In bacteriochlorophyll a the porphyrin ring is further reduced (7-8), and the group in position 3 is now a -COCH, group. In addition, in bacteriochlorophyll b, the group in position 8 is a = CHCH, group. 30 Cluster A number of metal centers grouped close together which can have direct metal bonding interactions or interactions through a bridging ligand, but are not necessarily held together by these interactions. Examples can be found under the entries r2Fe-25'1, r4Fe-45'1, ferredoxin, HiPIP, ironaulfur cluster, FeMo-cofactor, ferritin, metallo- thionein, nitrogenase, and Rieske iron-suwur protein. Cobalamin Vitamin B-12 or B12. A vitamin synthesized by microorganisms and conserved in animals in the liver. Deficiency or collective uptake of vitamin B-12 leads to pernicious anemia. Cobalamin is a substituted corrinCo(III) complex in which the cobalt atom is bound to the four nitrogen atoms of the corrin ring, an axial group R and 5,6dimethylbenzimidazole.The latter is linked to the cobalt by the N-3 nitrogen atom and is bound to the C-1 carbon of a ribose molecule by the N-1 nitrogen atom. Various forms of the vitamin are known with different R groups such as R = CN, cyanocobalamin; R = OH, hydroxocobalamin; R = CH3 methylcobalamin; R = 5'-deoxyadenosyl, coenzyme B-12. Coenzyme A low-molecular-weight, non-protein organic compound (often a nucleotide) 31 participating in enzymatic reactions as dissociable acceptor or donor of chemical groups or electrons. Cofactor An organic molecule or ion (usually a metal ion) that is required by an enzyme for its activity. It may be attached either loosely (coenzyme) or tightly (prosthetic group). Configuration In the context of stereochemistry, the term is restricted to the arrangements of atoms of a molecular entity in space that distinguishes stereoisomers, the isomerism between which is not due to conformational differences. Conformation The spacial arrangements of atoms affording distinction between stereoisomers which can be interconverted by rotations about formally single bonds. Cooperativity The phenomenon that binding of an effector molecule to a biological system either enhances or diminishes the binding of a successive molecule, of the same or different kind, to the same system. The system may be an entyme, or a protein that specifically binds another molecule such as oxygen or DNA. The effector molecule may be an enzyme substrate or an allosteric effector. The enzyme or protein exists in different conformations, with different catalytic rates or binding affinities, and binding of the effector molecule changes the proportion of 32 these conformations. Enhanced binding is named positive cooperativity; diminished binding is named negative cooperativity. A well-known example of positive cooperativity is in hemoglobin, In biocatalysis it was originally proposed that only multi-subunit enzymes could respond in this way. However single-subunit enzymes may give such a response (socalled mnemonic enzymes). Coordination A coordination entity is composed of a central atom, usually that of a metal, to which is attached a surrounding array of other atoms or group of atoms, each of which is called a ligand. A coordination entity may be a neutral molecole, a cation or an anion. The ligands may be viewed as neutral or ionic entities that are bonded to an appropriately charged central atom. It is standard practice to think of the ligand atoms that are directly attached to the central atom as defining a coordination polyhedron (tetrahedron, square plane, octahedron, etc.) about the central atom. The coordination number is defined as being equal to the number of sigma-bonds between ligands and the central atom; this definition is not necessarily appropriate in all areas of (coordination) chemistry. In a coordination formula, the central atom is listed first. The formally anionic ligands appear next and they are listed in alphabetic order according 33 to the first symbols of their formula. The neutral ligands follow, also in alphabetical order, according to the same principle. The formula for the entire coordination entity, whether charged or not, is enclosed in square brackets. In a coordination name, the ligands are listed in alphabetical order, without regard to charge, before the name of the central atom. Numerical prefixes indicating the number of ligands are not considered in determining that order. All anionic coordination entities take the ending -ate, whereas no distinguishing termination is used for cationic or neutral coordination entities. Crystal field 34 Crystal field theory is the theory which interprets the properties of coordination entities on the basis that the interaction of the ligands and the central atom is a strictly ionic or ion-dipole interaction resulting from electrostatic attractions between the central atom and the ligands. The ligands are regarded as point negative (or partially negative) charges surrounding a central atom; covalent bonding is completely neglected. The splitting or separation of energy levels of the five degenerate d orbitals in a transition metal, when the metal is surrounded by ligands arranged in a particular geometry with respect to the metal center, is called the crystal field splitting. See also ligandjield. C-terminal amino-acid residue See amino-acid residue. Cytochrome A heme protein that transfers electrons, and exhibits intense absorption bands (between 510 and 615 nm in the reduced form. Cytochromes are designated types a, b, c or d, depending on the type of heme. The iron undergoes oxidation-reduction between oxidation states Fe(II) and Fe(III). Most cytochromes are hemochromes, in which the fifth and sixth coordination sites in the iron are occupied by strong field ligands, regardless of the oxidation state of iron. Cytochrome-c oxidase An eniyme, ferrocytochrome-c: dioxygen oxidoreductase. The major respiratory protein of animal and plant mitochondria, it catalyzes the oxidation of Fe(II)-cytochrome c, and the reduction of dioxygen to water. Contains two hemes and three copper atoms, arranged in three centers. Heme and copper form a center that reacts with dioxygen; the second heme is cytochrome; the third site, copper, is a dinuclear center. Cytochrome P450 General term for a group of heme-containing monooxygenases . Named from the prominent absorption band of the Fe(II)carbonyl complex. The heme comprises protophorphyrin, and the proximal ligand to 35 iron is a cysteine sulfur. Cytochromes P-450 of microsomes in tissues such as liver are responsible for metabolism of many xenobiotics, including drugs. Others, such as the mitochondrial eniymes from adrenal glands, are involved in biosynthetic pathways such as those of steroids. The reaction with dioxygen appears to involve higher oxidation states of iron, such as Fe(IV)=O. Dehydrogenase An oxidoreductase which catalyzes the removal of hydrogen atoms from a substrate. Denitrification The reduction of nitrates to nitrites, nitrogen monoxide (nitric oxide), dinitrogen oxide (nitrous oxide) and ultimately dinitrogen catalyzed by microorganisms, e.g. facultative aerobic soil bacteria under anaerobic conditions. Denticity The number of donor groups from a given ligand attached to the same central atom. Deoxyribonucleic A high-molecular-mass linear polymer composed of nucleotides containing 2acid (DNA) deoxyribose and linked between positions 3' and 5' by phosphodiester groups; DNA contains the genetic information of organisms. The double-stranded form consists of a double helix of two complementary chains that run in opposite directions and are held together by hydrogen 36 bonds between pairs of the complementary nucleotides. The way the helices are constructed may differ and is usually designated as A, B, Z, etc. Occasionally, alternative structures are found, such as those with Hoogsteen base pairing. Desferrioxamine (dfo) Chelating agent used world-wide in the treatment of iron overload conditions, such as hemochromatosis and thalassemia. Diamagnetic Substances having a negative magnetic susceptibility are diamagnetic. They are repelled by a magnetic field. Dinuclear See nuclearity. Dioxygenase An enzyme that catalyzes the insertion of two oxygen atoms into a substrate, both oxygens being derived from O2. Dismutase An enzyme that catalyzes a disproportionation reaction. Dismutation See disproportionation. Disproportionati on Any chemical reaction of the type A + A + A' + A" where A, A' and A'' are different chemical species. The reverse of disproportionation (or dismutation) is called comproportionation. Dissociation See stability constant. 37 constant DNA See deoxyribonucleic acid. Domain An independently folded unit within a protein, often joined by a flexible segment of the polypeptide chain. Donor atom symbol A polydentate ligand possesses more than one donor site, some or all of which may be involved in coordination. To indicate the points of ligation, a system is needed. The general and systematic system for doing this is called the kappa convention: single ligating atom attachments of a polyatomic ligand to a coordination centre are indicated by the italic element symbol preceded by a Greek kappa, . In earlier practice, the different donors of the ligand were denoted by adding to the end of the name of the ligand the italicized symbol(s) for the atom or atoms through which attachment to the metal occurs. Electron transfer protein A protein, often containing a metal ion that oxidizes and reduces other molecules by means of electron transfer. Endogenous Originating internally. In the description of metal ion coordination in metalloproteins, endogenous refers to internal, or proteinderived, ligands. 38 Entatic state A state of an atom or group which due to its binding in a protein has its geometric or electronic condition adapted for function. Enzyme A macromolecule that functions as a biocatalyst by increasing the reaction rate, frequently containing or requiring one or more metal ions. In general, an enzyme catalyzes only one reaction type (reaction specificity) and operates on only a narrow range of substrates (substrate specificity). Substrate molecules are attacked at the same site (regiospecificity) and only one or preferentially one of the enantiomers of chiral substrate or of racemic mixtures is attacked (enantiospecificity). Equilibrium constant See acidity constant and stability constant. Exogenous Originating externally. In the context of metalloprotein ligands, exogenous describes ligands added from an external source, such as CO or O2. FeMo-cofactor An inorganic cluster that is found in the FeMo protein of the molybdenumnitrogenae and is essential for the catalytic reduction of N, to ammonia. This cluster contains Fe, Mo and S in a 7: 1 : 9 ratio. The structure of the cofactor within the FeMo protein can be described in terms of two cuboidal subunits, Fe,S, and MoFe,S, 39 bridged by three S2- ions and “anchored” to the protein by a histidine bound via an imidazole group to the Mo atom and by a cysteine bound via a deprotonated SH group to an Fe atom of the Fe,S, subunit. The Mo atom at the periphery of the molecule is sixcoordinate and in addition to the three sulfide-ligands and the histidine imidazole is also bound to two oxygen atoms from an (R)-homocitrate molecule. Fenton reaction Fe2+ + H2O2 Fe3+ + OH + OH-. This is the iron-salt-dependent decomposition of dihydrogen peroxide, generating the highly reactive hydroxyl radical, possibly via an oxoiron(1V) intermediate. Addition of a reducing agent such as ascorbate leads to a cycle which increases the damage to biological molecules. See also Haber-Weiss reaction. Ferredoxin A protein containing more than one iron and acid-labile sulfur, that displays electrontransfer activity but not classical enzyme function. See also HiPIP. 40 Ferriheme An iron(III) porphyrin coordination complex. Ferritin An iron storage protein consisting of a shell of 24 protein subunits, encapsulating up to 4500 iron atoms in the form of a hydrated iron(III) oxide. Ferrochelatase An enzyme which contains an Fe-S cluster and catalyzes the insertion of iron into protoporphyrin to form heme.. Ferroheme An iron(II) porphyrin coordination complex. Formation constant See stability constant. Gold drugs Gold coordination compounds used in the treatment of rheumatoid arthritis, examples being auranofin, (tetraacetylthioglucosatoS)(triethylphosphane)gold(I), and myocrysin, disodium thiomalatogold(I). Haber-Weiss reaction The Haber-Weiss cycle consists of the following two reactions : H2O2 + OH H2O + O2- + H+ and H2O2 + O2 O2 + OH + OHThe second reaction achieved notoriety as a possible source of hydroxyl radicals. However, it has a negligible rate constant. It is believed that iron(II1) complexes can 41 catalyze this reaction: first Fe(II1) is reduced by superoxide, followed by oxidation by dihydrogen peroxide. See also Fenton reaction. Hard acid A Lewis acid with an acceptor centre of low polarizability. It preferentially associates with hard bases rather than with soft bases, in a qualitative sense (sometimes called “HSAB rule”). Conversely a soft acid possesses an acceptor centre of high polarizability and exhibits the reverse preference for a partner for coordination. Hard base A Lewis base with a donor centre of low polarizability; the converse applies to soft bases. See also hard acid. Heme A near-planar coordination complex obtained from iron and the dianionic form of porphyrin. Derivatives are known with substituents at various positions on the ring named a, b, c, d, etc. Heme b, derived from protoporphyrin, is the most frequently occurring heme. Hemocyanin A dioxygen-carrying protein (from invertebrates, e.g. arthropods and molluscs), containing dinuclear type 3 copper sites. Hemoglobin A dioxygen-carrying heme protein of red blood cells, generally consisting of two alpha and two beta subunits, each 42 containing one molecule of protoporphyrin. High-spin See low-spin. HiPlP Formerly-used abbreviation for HighPotential Iron-sulfur Protein, now classed as a ferredoxin. An electron transfer protein from photosynthetic and other bacteria, containing a [4Fe-4S] cluster which undergoes oxidation-reduction between the [4Fe-4S]2+ and [4Fe-4S]3+ states. Hydration Addition of water or the elements of water (i.e. H and OH) to a molecular entity. The term is also used in a more restricted sense for the process: A (gas) A (aqueous solution); cf. the use of the term in inorganic and physical chemistry to describe the state of ions of an aqueous electrolyte solution. See also aquation and salvation. Hydrogenase An envme, dihydrogen:acceptor oxidoreductase, that catalyzes the formation or oxidation of H2. Hydrogenases are of various types. One class ([Fe]-hydrogenases) contains only iron-sulfur clusters. The other major class ([NiFe]-hydrogenases) has a nickel-containing center and iron-sulk clusters; a variation of the latter type ([NiFeSe]-hydrogenases) contains selenocysteine. 43 Hydrolase An enqvme of EC class 3, also known as a hydro-lyase, that catalyzes the hydrolysis of a substrate. Hydrolysis Solvolysis by water. Hydrophilic The term is used to mean "water preferring". May also be used to describe the character of interaction of a particular atomic group with the medium. Hydrophobic interaction The tendency of hydrocarbons (or of lipophilic hydrocarbon-like groups in solutes) to form intermolecular aggregates in an aqueous medium, and analogous intramolecular interactions. The name arises from the attribution of the phenomenon to the apparent repulsion between water and hydrocarbons. Use of the misleading alternative term hydrophobic bond is discouraged. Inhibition The decrease in the rate of a reaction brought about by the addition of a substance (inhibitor). Intercalation compounds Compounds resulting from inclusion, usually without covalent bonding, of one kind of molecule (the guest molecule) in a matrix of another compound (the host compound), which has a layered structure. The host compound, with a rather rigid structure, may be macromolecular, 44 crystalline, or amorphous. Iron-responsive element A specific base sequence in certain messenger RNAs that code for various proteins of iron metabolism, which allows regulation at translational level by the ironreponsive protein. Iron-responsive protein (IRP) A protein that responds to the level of iron in the cell, and regulates the biosynthesis of proteins of iron metabolism, by binding to the iron-responsive element on messenger RNA. Iron-sulfur proteins Proteins in which non-heme iron is coordinated with cysteine sulfur and usually also with inorganic sulfur. Divided into three major categories: rubredoxins; "simple ironsulfur proteins", containing only ironsurfur clusters, and "complex iron-sulfur proteins", containing additional active redox centers such as flavin, molybdenum or heme. In most iron-sulfur proteins the clusters function as electron transfer groups, but in others they have other functions such as catalysis of hydrataseldehydratase reactions, maintenance of protein structure, or regulation of activity. lsomerase An enzyme of EC class 5, which catalyzes the isomerization of a substrate. Labile The term has loosely been used to describe 45 either a relatively unstable and transient chemical species or a relatively stable but reactive species. Lewis acid A molecular entity that is an electron pair acceptor and therefore able to react with a Lewis base to form a Lewis adduct, by sharing the electron pair furnished by the Lewis base. Lewis adduct The adduct formed between a Lewis acid and a Lewis base. Lewis base A molecular entity able to provide a pair of electrons and thus capable of coordination to a Lewis acid, thereby producing a Lewis adduct. Ligand In inorganic chemistry the ligands are the atoms or groups of atoms bound to the central atom (see also coordination). The root of the word is sometimes converted into the verb to ligate, meaning to coordinate as a ligand, and the derived participles, ligating and ligated. This use should not be confused with its use to describe the action of ligases (a class of enzymes). The names for anionic ligands, whether inorganic or organic, end in -O. In general, if the anion name ends in ide, or -ate, the final -e is replaced by -O, giving ido, and -ato, respectively. Neutral and cationic ligand names are used without modification. Ligands bonded by a single 46 carbon atom to metals are regarded as radical substituents, their names being derived from the parent hydrocarbon, from which one hydrogen atom has been removed. In general, the final letter -e of the name is replaced by -yl. In biochemistry the term ligand has been used more widely: if it is possible or convenient to regard part of a polyatomic molecular entity as central, then the atoms or groups or molecules bound to that part may be called ligands. Ligand field Ligand field theory is a modified crystal field theory that assigns certain parameters as variables rather than taking them as equal to the values found for free ions, thereby taking into account the potential covalent character of the metal-ligand bond. Low-spin In any coordination entity with a particular dn (1 < n < 9) configuration and a particular geometry, if the n electrons are distributed so that they occupy the lowest possible energy levels, the entity is a low-spin complex. If some of the higher energy d orbitals are occupied before all the lower energy ones are completely filled, then the entity is a high-spin complex. Metabolism The entire physical and chemical processes involved in the maintenance and reproduction of life in which nutrients are broken down to generate energy and to give 47 simpler molecules (catabolism) which by themselves may be used to form more complex molecules (anabolism). Metalloenzyme An enzyme that, in the active state, contains one or more metal ions which are essential for its biological function. Mixed valency This is one of several names, such as ‘mixed oxidation state’ or ‘non-integral oxidation state’ used to describe coordination compounds and clusters in which a metal is present in more than one level of oxidation. The importance in biology is due to the often complete delocalization of the valence electrons over the cluster, allowing efficient electron-transfer processes. See also oxidation number. Model A synthetic coordination entity that closely approaches the properties of a metal ion in a protein and yields useful information concerning biological structure and function. Given the fact that the term is also loosely used to describe various types of molecular structures, constructed, for example, in the computer, the term biomimetic is more appropriate. Monooxygenase An enzyme that catalyzes the insertion of one atom of oxygen, derived from O,, into an aromatic or aliphatic compound. The reaction is coupled to the oxidation of a co- 48 substrate such as NAD(P)H or 2oxoglutarate. Motif A pattern of amino acids in a protein sequence which has a specific function, e.g. metal binding. See also consensus sequence. Multicopper oxidases A group of enzymes that oxidize organic substrates and reduce dioxygen to water. These contain a combination of copper ions with different spectral features; called type I centers, type 2 centers, and type 3 centers, where the type 2 and type 3 sites are clustered together as a trinuclear unit. Wellknown examples are: laccase, ascorbate oxidase and ceruloplasmin. Myocrysin See gold drugs. Myoglobin A monomeric dioxygen-binding heme protein of muscle tissue, structurally similar to a subunit of hemoglobin. Nitrate reductase A metalloenzyme, containing molybdenum that reduces nitrate to nitrite. Nitrite reductase A metalloenzyme that reduces nitrite. Dissimilatory nitrite reductases contain copper and reduce nitrite to nitrogen monoxide. Assimilatory nitrite reductases contain siroheme and iron-sulfur clusters and reduce nitrite to ammonia. 49 Nitrogenase An enzyme complex (EC 1.18.6.1) from bacteria that catalyzes the reduction of dinitrogen to ammonia: N2 + 8e- + 10H+ 2NH4+ + H2 with the simultaneous hydrolysis of at least 16 ATP molecules. The electron donor is reduced ferredoxin or flavodoxin. Dihydrogen is always a co-product of the reaction. Ethyne (acetylene) can also be reduced to ethene (ethylene) and in some cases ethane. All nitrogenases are ironsulfur proteins. Three different types which differ in the type of cofactor present have been identified: molybdenum-nitrogenase (the most common, which contains the ironmolybdenum cofactor), vanadiumnitrogenase, and iron-only nitrogenase. See also FeMo-cofactor. Nitrogen fixation The assimilation of dinitrogen by microbial reduction to ammonia and conversion into organonitrogen compounds such as amino acids. Only a limited number of microorganisms are able to fix nitrogen. See also nitrogenase. N-terminal amino-acid residue See amino-acid residue. Nuclearity The number of central atoms joined in a single coordination entity by bridging 50 ligands or metal-metal bonds is indicated by dinuclear, trinuclear, tetranuclear, polynuclear, etc. Nucleic acids Macromolecules composed of sequences of nucleotides that perform several functions in living cells, e.g. the storage of genetic information and its transfer from one generation to the next (DNA), and the expression of this information in protein synthesis (mRNA, tRNA), and may act as functional components of subcellular units such as ribosomes (rRNA). RNA contains D-ribose, DNA contains 2-deoxy-D-ribose as the sugar component. Currently, synthetic nucleic acids can be made consisting of hundreds of nucleotides. See also oligonucleotide. Nucleosides Compounds in which a purine or pyrimidine base is -N-glycosidically bound to C-1 of either 2-deoxy-D-ribose or of D-ribose, but without any phosphate groups. The common nucleosides in biological systems are adenosine, guanosine, cytidine, and uridine (which contain ribose) and deoxyadenosine, deoxyguanosine, deoxycytidine and thymidine (which contain deoxyribose). Nucleotides Nucleosides with one or more phosphate groups esterified mainly to the 3’- or the 5’position of the sugar moiety. Nucleotides found in cells are adenylic acid, guanylic 51 acid, uridylic acid, cytidylic acid, deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid and thymidylic acid. See also adenosine 5 triphosphate, NAD+, NADP. Oxidase An enzyme which catalyzes the oxidation of substrates by O2. Oxidation number The oxidation number of an element in any chemical entity is the number of charges which would remain on a given atom if the pairs of electrons in each bond to that atom were assigned to the more electronegative member of the bond pair. The oxidation (Stock) number of an element is indicated by a roman numeral placed in parentheses immediately following the name (modified if necessary by an appropriate ending) of the element to which it refers. The oxidation number may be positive, negative or zero. Zero, not a roman numeral, is represented by the usual cipher, 0. The positive sign is never used. An oxidation number is always positive unless the minus sign is explicitly used. Note that it cannot be nonintegral (see also mixed valency). Non-integral numbers may seem appropriate in some cases where a charge is spread over more than one atom, but such a use is not encouraged. In such ambiguous cases, the charge number, which designates ionic charge, can be used. A charge (Ewens-Bassett) number is a number 52 in parentheses written without a space immediately after the name of an ion, and whose magnitude is the ionic charge. Thus the number may refer to cations or anions, but never to neutral species. The charge is written in arabic numerals and followed by the sign of the charge. In a coordination entity, the oxidation number of the central atom is defined as the charge it would bear if all the ligands were removed along with the electron pairs that were shared with the central atom. Neutral ligands are formally removed in their closed-shell configurations. Where it is not feasible or reasonable to define an oxidation state for each individual member of a group or cluster, it is again recommended that the overall oxidation level of the group be defined by a formal ionic charge, the net charge on the coordination entity. Paramagnetic Substances having a positive magnetic susceptibility are paramagnetic. They are attracted by a magnetic field. Peroxidase A heme protein (donor: hydrogen peroxide oxidoreductase, EC class 1.1 1.1) which catalyzes the oneelectron oxidation of a substrate by dihydrogen peroxide. Substrates for different peroxidases include various organic compounds, cytochrome C, halides, and Mn2+. 53 Photosynthesis A metabolic process in plants and certain bacteria, using light energy absorbed by chlorophyll and other photosynthetic pigments for the reduction of CO2 followed by the formation of organic compounds. Plastocyanin An electron transfer protein, containing a type 1 copper site, involved in plant and cyanobacterial photosynthesis, which transfers electrons to Photosystem I. Polydentate See chelation, donor atom symbol. Porphyrin A macrocyclic molecule that contains four pyrrole rings linked between the alpha positions of the pyrrole rings. Porphyrins coordinated to a metal ion. Primary structure The amino-acid sequence of a protein or nucleotide sequence of DNA or RNA. Protoporphyrin The porphyrin ligand of heme b. Heme b is a Fe(II) porphyrin complex readily isolated 54 from the hemoglobin of beef blood, but also found in other proteins including other hemoglobins, myoglobins, cytochromes P450, catalases, peroxidases as well as b type cytochromes. Protoporphyrin IX contains four methyl groups in positions 2, 7, 12 and 18, two vinyl groups in positions 3 and 8 and two propionic acid groups in positions 13 and 17. Quaternary structure The level of structural organization in oligomeric proteins (i.e., those composed of more than one subunit) represented by the number and arrangement of the subunits and the interactions between them. Radical A molecular entity possessing one or more unpaired electrons, formerly often called “free radical”. A radical may be charged, positively (radical cation) or negatively (radical anion). Paramagnetic metal ions are not normally regarded as radicals. Redox potential Any oxidation-reduction (redox) reaction can be divided into two half reactions: one in which a chemical species undergoes oxidation and one in which another chemical species undergoes reduction. If a halfreaction is written as a reduction, the driving force is the reduction potential. If the half-reaction is written as oxidation, the driving force is the oxidation potential related to the reduction potential by a sign 55 change. So the redox potential is the reductiodoxidation potential of a compound measured under standard conditions against a standard reference half-cell. In biological systems the standard redox potential is defined at pH = 7.0 versus the hydrogen electrode and partial pressure of hydrogen = 1 bar. Ribonucleic acids Linear polymer molecules composed of a chain of ribose units linked between (RNA) positions 3 and 5 by phosphodiester groups. The bases adenine or guanine (via atom N-9) or uracil or cytosine (via atom N-l), respectively, are attached to ribose at its atom C-1 by -N-glycosidic bonds (see nucleotides). The three most important types of RNAs in the cell are: messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA). Rieske ironsulfur protein An iron-sulfur protein of the mitochondria1 respiratory chain, in which the (2Fe-2S] cluster is coordinated to two sulfur ligands from cysteine and two imidazole ligands from histidine. The term is also applied to similar proteins isolated from photosynthetic organisms and microorganisms, and other proteins containing [2Fe-2S] clusters with similar coordination. Rubredoxin An iron-sulfur protein without acid-labile 56 sulfur, in which an iron center is coordinated by four sulfur containing ligands, usually cysteine. The function, where known, is as an electron carrier. Secondary structure Level of structural organization in proteins described by the folding of the polypeptide chain into structural motifs such as alpha helices and beta sheets, which involve hydrogen bonding of backbone atoms. Secondary structure is also formed in nucleic acids, especially in single-stranded RNA’s by internal base pairing. Sequence The order of neighbouring amino acids in a protein or the purine and pyrimidine bases in RNA or DNA. See also primary structure. Soft acid See hard acid. Soft base See hard base. Solvation Any stabilizing interaction of a solute (or solute moiety) and the solvent or a similar interaction with solvent of groups of an insoluble material (i.e., the ionic groups of an ion-exchange resin). They generally involve electrostatic forces and Van der Waals forces, as well as chemically more specific effects such as hydrogen bond formation. Solvolysis Reaction with a solvent involving the 57 rupture of one or more bonds in the reacting solute. Stability constant An equilibrium constant that expresses the propensity of a species to form from its component parts. The larger the stability constant, the more stable is the species. The stability constant (formation constant) is the reciprocal of the instability constant (dissociation constant). Stable Stable is a term describing a system in a state of equilibrium corresponding to a local minimum of the appropriate thermodynamic potential for the specified constraints on the system. Stability cannot be defined in an absolute sense, but if several states are in principle accessible to the system under given conditions, that with the lowest potential is called the stable state, while the other states are described as metastable. Unstable states are not at a local minimum. Transitions between metastable and stable states occur at rates which depend on the magnitude of the appropriate activation energy barriers which separate them. Stereochemical Refers to the three-dimensional view of a molecule either as such or in a projection. Stock number See oxidation number. Substrates (1) A chemical species of particular interest, 58 the reaction of which with some other chemical reagent is under observation (e.g. a compound that is transformed under the influence of a catalyst). (2) The chemical entity whose conversion to a product or products is catalyzed by enzymes. (3) A solution or dry mixture containing all ingredients which are necessary for the growth of a microbial culture or for product formation. (4) A component in the nutrient medium, supplying the organisms with carbon (Csubstrate), nitrogen (N- substrate) etc. Superoxide dismutases (SOD) Enzymes which catalyze the dismutation reaction of superoxide anion to dihydrogen peroxide and dioxygen. The enzymes have active sites containing either copper and zinc (Cd/Zn superoxide dismutase), or iron (Fesuperoxide dismutase), or manganese (Mnsuperoxide dismutase). Tertiary structure The overall three-dimensional structure of a biopolymer. For proteins this involves the side chain interactions and packing of secondary structure motif. For nucleic acids this may be the packing of stemloops or supercoiling of double helices. Trace elements Elements required for physiological functions in very small amounts that vary for 59 different organisms. Included among the trace elements are Co, Cu, F, Fe, I, Mn, Mo, Ni, Se, V, W, and Zn. Excess mineral intake may produce toxic symptoms. Transferrin An iron-transport protein of blood plasma, which comprises two similar iron-binding domains with high affinity for Fe(III). Similar proteins are found in milk (lactoferrin) and eggs (ovotransferrin). Transition element Transition element is an element whose atom has an incomplete d-sub-shell, or which gives rise to a cation or cations with an incomplete d-sub-shell. The First Transition Series of elements is Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu. The Second and Third Transition Series are similarly derived : these include the lanthanoids (lanthanides) and actinoids (actinides) respectively which are designated inner (or f ) transition elements of their respective Periods in the Periodic Table. Type 1, 2, 3 copper Different classes of copper-binding sites in proteins, classified by their spectroscopic properties as Cu(II). In type 1, or blue copper centers the copper is coordinated to at least two imidazole nitrogens from histidine and one sulfur from cysteine. They are characterized by small copper hyperfine couplings and a strong visible absorption in the Cu(II) state. In type 2, or non-blue 60 copper sites, the copper is mainly bound to imidazole nitrogens from histidine. Type 3 copper centers comprise two spin-coupled copper ions, bound to imidazole nitrogens. Zinc finger A domain, found originally in certain DNAbinding proteins and subsequently in other classes of proteins, comprising a helix-loop structure in which a zinc ion is coordinated to 2-4 cysteine sulfurs, the remaining ligands being histidines. In many proteins of this type the domain is repeated several times. Zwitterionic compound A neutral compound having electrical charges of opposite sign, delocalized or not, on adjacent or nonadjacent atoms. 61 62 LECTURE NOTES 63 64 CHAPTER I Introduction to Bioinorganic Chemistry Bioinorganic chemistry is a leading discipline at the interface of chemistry and biology. A lot of biological processes require metal ions. Among them, there are metabolism, nitrogen and oxygen fixation, photosynthesis, nerve transmission, and many others. Metals are being introduced into living bodies as diagnostic probes and drugs. Thus, bioinorganic chemistry is a field that examines the role of metals in biology. Bioinorganic chemistry includes the study of both natural phenomena such as the behavior of metalloproteins as well artificially introduced metals, including those that are non-essential, in medicine and toxicology. Bioinorganic chemistry is important in realizing the implications of electron-transfer proteins, substrate bindings and activation, atom and group transfer chemistry, as well as metal properties in biological chemistry. Major element content in living bodies differ from those in the Earth’ rust but is in a good correlation with that in the sea water: 65 Chemical elements essential to life forms can be divided into the following: (i) Bulk elements: C, H, N, O, P, S; (ii) Macrominerals and ions: Na+, K+, Mg2+, Ca2+, Cl-, PO43-, SO42-; (iii) Trace elements: Fe2+/3+, Zn2+, Cu2+; (iv) Ultratrace elements comprises of: (a) non-metals: F, I, Se, Si, As, B (b) metals: Mn, Mo, Co, Cr, V, Ni, Cd, Sn, Pb, Li. About 99% of mammals' mass are the elements carbon, nitrogen, calcium, sodium, chlorine, potassium, hydrogen, phosphorus, oxygen and sulfur. The organic compounds (proteins, lipids and carbohydrates) contain the majority of the carbon and nitrogen and most of the oxygen and hydrogen is present as water. The entire 66 collection of metal-containing biomolecules in a cell is called the metallome. The biological functions of some of the metals are presented in the Table 1. Table 1. Biological functions of some metals. The essentiality of metallic elements is defined by the following: (1) A physiological deficiency appears when the element is removed from the diet. (2) The deficiency is relieved by the addition of that element to the diet. (3) A specific biological function is associated with the element. Some applications of medicinal relevant elements are listed bellow (see Table 2). 67 Table 2. Medicinal applications of some metal cations Metal ion or salt Application + Li Treatment of bipolar disorder (maniac depression) and hypertension 3+ Gd Contrast agent in magnetic resonance tomography of soft tissues BaSO4 Contrast agent for X-ray tomography. Sun protection 99m Tc (a metastable γ-emitter; t1/2 = 6 h): Radio diagnostics (bone cancer, infarct risk, …) 2+ Pt Chemotherapy (e.g. with cisplatin cis[Pt(NH3)2Cl2]) of cancer (ovaria, testes) + Au Therapy of rheumatic arthritis Sb3+ Treatment of inflammatory skin pimples like acne Bi3+ Treatment of gastritis Transition metal ions commonly are not present in a free form, but rather coordinated (complexed) to ligands. In particular, this applies to metal ions in the active centres of enzymes or otherwise integrated into peptides and proteins. Examples for the respective ligands are listed below (N-functional: peptide moiety, porphinogenes, histidine; O-functional: tyrosinate, serinate, glutamate and aspartate; S-functional: cysteinate and methionine): 68 Some additional inorganic ligands are also present: There are three major avenues of investigation in bioinorganic chemistry. The first involves direct study of the structure and function of ‘metallobiomolecules’, an area which is traditionally that of a biochemist. The second major avenue involves an indirect approach, commonly the domain of the inorganic or organic chemists. The third involves the addition of metal ions or complexes as probes to biochemical structure and function. 69 Basic concepts of the method of molecular orbitals (МО) in description of covalent and coordinate bonds. According to МО method, the molecule consists of a set of nuclei and electrons disposed on molecular orbitals. Main tenets of the MO method: 1. Each electron in a molecule occupies a certain energetic level (molecular orbital, MO) which is characterised by a molecular -function and a corresponding set of quantum numbers. 2. The total number of formed МО equals the number of initial atomic orbitals. 3. Filling of MO occurs according to all the principles presented for atomic orbitals. 4. МО it is considered as a linear combination of atomic orbitals (MO – LCAO). Let's consider, for example, formation of molecule АВ. Valence electrons of each atom are on p-orbitals. If the wave function of the isolated atom A is А, and for the atom B it is В, so according to the МО method: ±АВ=С1А±С2В where С1 and С2 are coefficients considering the income of each atom in formation of a molecular orbital. 70 А _ E (ab*.) В А В + А В (b) The new molecular orbital with a lower energy (b) is known as a bonding orbital. As its energy is lower than the energy of an atomic orbital, electrons on it stabilize the molecule. The MO with a higher energy (ab*) is called an antibonding orbital. Electrons on it tend to destroy the molecule. Stability of a molecule is described by a bond order, B.O. B.O. = ½ (No of bonding electrons – No of antibonding electrons) If B.O. = 0 the number of electrons on binding orbitals equals the number of electrons on antibinding electrons. Such molecule is unstable and breaks up to initial atoms (does not exist). Conditions for formation of MO from AO are the following: (i) close values of energies of overlapping atomic orbitals; (ii) considerable overlap of AO (formation of and -types of MO); (iii) identical spatial disposition of AO (рх - рх, instead of рх - рz). On level power molecular orbitals two-nuclear molecules settles down in a following order: 71 s(1s)s(1s)s(2s)s*(2s)x(2px)z(2pz)=y(2py)z*(2pz) =y*(2py)x*(2px) Examples of the description of molecules using the MO method (energy diagrams of molecules and molecular ions): 72 73 74 75 76 Coordination compounds (definition). Coordination compounds, or complexes, are integral molecular or ionic units consisting of a central metal ion (or atom), bonded to a defined number of ligands in a defined geometrical arrangement. The ligands can be ions or (induced) dipolar molecules. Each ligand provides a free electron pair, i.e. the ligands are Lewis bases, while the metal in the coordination centre is the Lewis acid. The bonding can thus be described in terms of Lewis acid/Lewis base interaction. Other descriptions of the bonding situation are: (i) donor bond; (ii) coordinative covalent bond, often denoted by L→M, where L = ligand and M = metal. Complexes tend to be stable when the overall electron configuration at the metal centre (the sum of metal valence electrons plus electron pairs provided by the ligands) is 18 (or 16 for the late transition metals). Structure of complex compounds In a molecule of a complex compound, one of the atoms, generally positively charged, occupies the central site (central ion or complexing agent). Oppositely charged ions or neutral molecules called ligands are coordinated around the central ion. The complexing agent and ligands form inner sphere of a complex compound. It is characterized by coordinate bonds which are formed while overlapping of empty p- and d-orbitals of a central ion and orbitals containing lone electron pairs of ligands. The ions in the outer sphere are mainly bonded to the complex ions by forces of electrostatic interaction (ionic bonds). The total number of coordinate bonds formed by the complexing agent is known as coordination number of the central ion. It mainly depends upon the charge of the complexing agent (for monocharged ions it usually equals 1, for discharged 77 ions - 4 or 6, for tricharged - 6 and above), and the size of an ion (the larger the central ion, the greater its coordination number is, for lanthanides and actinides it can reach to 12).. Ligands possess the property of dentacity. In accordance with the number of coordinate bonds formed by a ligand with the central ion, the ligand may be a monodentate, bidentate, or polydentate. Dentacity is defined by number lone electronic pairs in a molecule of a ligand and their mutual spatial disposition. For example, the ammonia molecule NH3 has one lone electronic pair belonging to the N-atom therefore ammonia is a monodentate ligand. The water molecule has two lone electronic pairs, and chloride-ion has them four. However because valence orbitals of oxygen and chlorine are in the sp3-type of hybridization and are located under a corner 10928 ’ they cannot form chemical bonds with the same central ion, therefore such лиганды are monodentate except the case of polynuclear complexes where they act as bidentate bridging ligands. Example: Na2 [Cu2Cl6] Cl Na2 Cl Cl Cu Cu Cl Cl Cl Dicharged acidic anions such as SO32–, C2O42– ets. are usually bidentate chelating ligands. Example: Na2[Cd(SO3)2] 78 O Na2 O S O Cd O S O O Properties Multidentate ligands are much stronger complexes than monodentate ligands; Chelates remain stable even at very dilute concentrations (less dissociation); Chelate effect: increase of entropy G = H – TS, H for multi- and monodentate complexes Cu(H2O)42+ + 4NH3 [Cu(NH3)4]2+ + 4H2O Cu(H2O)42+ + N4X [Cu(N4X)]2+ + 4H2O Chelate therapy (detoxification). 79 Electronic structure of complex ions. I. Valence bond theory (Pauling). Interaction of lone electronic pairs of ligands with empty valence orbitals of the central ion of different types leads to their hybridization. For example, the electronic structure of a complex ion [Cu(NH3)4]2+ can be reflected as following: 2 2 6 2 6 10 1 29Cu: 1s 2s 2p 3s 3p 3d 4s 2+ 2 2 6 2 6 9 0 0 0 29Cu : 1s 2s 2p 3s 3p 3d 4s 4p 4d 3d 4s 4p 4d 4 :NH3 Interaction of one s- and three p-orbitals leads to the sp3hybridization of the central ion (tetrahedral complex). Entering of lone electronic pairs of ligands into valence orbitals of the central ion leads to their interaction with the electrons of 3d-orbitals. This interaction is defined by degree of penetration of electrons of ligands on empty orbitals of metallic cations. In connection of force of interaction, ligands may be arranged in a spectrochemical series and are devided into ligands of weak and strong field: CO, CN– > NO2– > NH3 Ligands of a strong field > SCN– > H2O > OH–- > F– Ligands of a weak field > Cl– Lone electronic pairs of ligands of a strong field deeply enter the valence electronic shell of the central ion and cause pairing of electrons of the 3d-subshell. 80 As a result, intra-orbital low-spin complexes are formed (coordination bonds are formed with participation of internal 3dorbitals, the formed complex has no or few unpaired electrons): 2 2 6 2 6 7 2 27Co: 1s 2s 2p 3s 3p 3d 4s 3+ 2 2 6 2 6 6 0 0 0 27Co : 1s 2s 2p 3s 3p 3d 4s 4p 4d 3d 4s 4p 4d 4s 4p 4d [Co(NH3)6]3+ 3d 6 :NH3 d2sp3-hybridization, octahedral complex. Lone electronic pairs of ligands of a weak field slightly interact with 3d-electrons of the central ion and do not cause their pairing. As a result, outer-orbital high-spin complexes are formed: 81 [CoF6]3– 3d 4s 4p 4d 6 :F- sp3d2-hybridization, octahedral complex. Crystal Field Theory (Electrostatic guide to splitting of d-levels). Ligand field splitting 0, LFSE (Ligand Field Splitting Energy); Spectrichemical series (0 increases): I- < Br- < S2- < Cl- < NO3- < OH- < H2O < NH3 < en < NO2- < CN- < CO; Metal 0 increases with increasing oxidation number and down a group: Mn(II) < Ni(II) < Fe(II) < V(II) < Fe(III) < Co(III) < Mn(IV) < Mo(III) < Pd(IV) < Pt(IV); Electronic configuration of a central atom depends on LFSE and P (pairing energy); Jahn Teller distortion: remove of degeneracy, increase of LFSE: i) splitting eg (dz2 lower energy) and t2g (dxy higher energy; ii) square coordination: dxy higher energy than dz2. 82 As an example, the versatility of iron as a function of its environment is is discussed. The ligand field can strongly alter the structural and ligand exchange properties of the metal ion (Figure 1). The ligand field can also alter the redox properties. For high-spin ferric ion, as found in the aquo complex or in many other complexes (including the class of microbial iron-transport agents called siderophores, to be discussed later), the coordination geometry is octahedral or pseudo-octahedral. Figure 1. Versatility of Fe coordination compounds. 83 In the relatively weak ligand field (high-spin ground state), the complex is highly labile. In a strong ligand field, such as an axially ligated porphyrin complex of ferric ion, or the simple example of the ferrocyanide anion, the low-spin complex is exchange-inert. Similarly, the high-spin octahedral ferrous complexes are exchange-labile, but the corresponding axially ligated porphyrin complexes, or the ferrocyanide complexes, are spin-paired (diamagnetic) and ligand exchange-inert. Large, bulky ligands or constrained ligands, such as those provided by metalloprotein and enzyme sites, can cause a tetrahedral environment, in which both ferrous ion and ferric ion form highspin complexes. Example: FeII (d6): In an octahedral (Oh) field, the degeneracy of the five d-orbitals is lifted. Depending on the strength of the ligand field, the ligand field stabilisation energy (i.e. the energy set free as all of the electrons are accommodated in the orbitals of lower energy) can be (i) less and (ii) more than the energy needed for electron pairing. In the case of (i), i.e. aqua ligands, a high-spin complex is formed; in the case of (ii), i.e. cyanido ligands, a low-spin complex is formed. Asymmetrically occupied orbital sets, as in the case of [Fe(H2O)6]2+, result in further stabilisation through symmetry lowering: Jahn Teller distortion: 84 85 Pearson’s concept of hard and soft interactions. Lewis concept: metal ions are acids because they accept electrons. Ligands are bases because they donate electrons. Hard acids tend to form complexes with hard bases (ionic bonds) Soft acids tend to form complexes with soft bases (covalent bonds) Hard acids: H+, Li+(> Na+…), Cr6+(> Cr3+) Intermediate acids: Fe2+, Mn2+, Cu2+, Zn2+ Soft acids: Au+(> Ag+, Cu+), Hg2+, Pt2+ Hard bases: F-, OH-, NH3, PO43-(> HPO42-), MoO42Intermediate bases: ClSoft bases: I-(> Br-…), S2-(> HS-, > H2S), AsS2- Chelate effect (Stabilisation of complexes by multidentate ligands). The chelate effect is an entropic effect (high entropy = high disorder [increase of particle number]). Example: The complex formed between the siderophore enterobactin: 86 (ent6-, a hexadentate ligand) and Fe3+ is particularly stable: [Fe(H2O)6]3+ + ent6- → [Fe(ent)]3- + 6H2O. 87 CHAPTER II. Transition Metal Chemistry Living organisms store and transport transition metals both to provide appropriate concentrations of them for use in metalloproteins or cofactors and to protect themselves against the toxic effects of metal excesses; metalloproteins and metal cofactors are found in plants, animals, and microorganisms. The form of the metals in living bodies is always ionic, but the oxidation state can vary, depending on biological needs. Transition metals for which biological storage and transport are significant are, in order of decreasing abundance in living organisms: iron, zinc, copper, molybdenum, cobalt, chromium, vanadium, and nickel. Iron Iron takes over a central role in biological events. On the one hand, this is due to its general availability (iron is abundant and ubiquitous in the geo- and biospheres), on the other hand, iron has specific and “biologically suitable” properties otherwise not (or less) available with other transition metals: (1) Ease of change between the oxidation states +II and +III (and disposability also of the oxidation states +IV and +V); (2) Formation of hexaaqua cations in water; these hexaaqua cations are Brønsted acids; (3) Tendency to form oligo- and polymers by condensation; (4) Easy change between high- and low-spin states in ligand fields of medium strength (spin cross-over); 88 (5) Flexibility with respect to the nature of the donating ligand function, the coordination number and coordination geometry. In aqueous solutions, the redox potential for the pair Fe /Fe at pH = 7 demonstrates that FeII is easily oxidised to FeIII under aerobic conditions: Fe2+ Fe3+ + e-; E = -0.23 V (at pH 7) 2+ 3+ Hexaaquairon(III) ions are cationic Brønstedt acids: [Fe(H2O)6]3+ + H2O [Fe(H2O)5OH]2+ + H3O+ pKS1 = 2.2 [Fe(H2O)5(OH)]2+ + H2O [Fe(H2O)4(OH)2]+ + H3O+ pKS2 = 3.5 [Fe(H2O)4(OH)2]+ + H2O [Fe(H2O)3(OH)3] (= Fe(OH)3·aq + H3O+) pKS3 = 6.0 The formation of ferric hydroxide Fe(OH)3·aq hence already starts in weakly acidic media. The protolytic reactions are accompanied by condensation reactions, leading to hydroxidoand oxido-bridged aggregates and finally to colloids and hardly soluble ferric oxide hydrates: 89 While coordination, Fe-ions posses the affinity to Odonating groups of the catecholate and hydroximate-like ligands: 90 The formation of coordinate bonds with N- and Sdonating atoms occurs in hemoglobine (a), cytochrom c (b) and rubredoxin (c) structures: 91 (a) (b) 92 (c) The distribution of specific iron complexes in living organisms depends strongly on function. For example, although there are many different iron complexes in the average human, the relative amounts of each type differ more than 650-fold (Table 3). The total amount of iron in humans is quite large, averaging more than three and up to five grams for a healthy adult. Most of the iron is present as hemoglobin, the plasma oxygen-transport protein. A much smaller amount of iron is present in myoglobin, a muscle oxygen-storage protein. Other examples of iron-containing proteins and their functions are included in Table 3. 93 Table 3. Average human Fe distribution Different iron coordination environments significantly changes the redox-potential of the Fe3+/Fe2+ redox-pair (Table 4). Table 4. Fe redox potentials 94 Chemical properties of zinc, copper, vanadium, chromium, molybdenum, and cobalt For zinc there is only the +2 oxidation state, and the hydrolysis of this ion is not a limiting feature of its solubility or transport. Zinc is an essential element for both animals and plants.The Zn2+ cation is an efficient Lewis acid, making it a useful catalytic agent in hydroxylation and other enzymatic reactions. The metal also has a flexible coordination geometry, which allows proteins using it to rapidly shift conformations to perform biological reactions. Two examples of zinc-containing enzymes are carbonic anhydrase and carboxypeptidase, which are vital to the processes of carbon dioxide (CO2) regulation and digestion of proteins, respectively. In vertebrate blood, carbonic anhydrase converts CO2 into bicarbonate and the same enzyme transforms the bicarbonate back into CO2 for exhalation through the lungs. Without this enzyme, this conversion would occur about one million times slower at the normal blood pH of 7 or would require a pH of 10 or more. The non-related β-carbonic anhydrase is required in plants for leaf formation, the synthesis of indole acetic acid (auxin) and alcoholic fermentation. Zinc serves a purely structural role in zinc fingers, twists and clusters (Figure 2). 95 Figure 2. Zinc fingers help read DNA sequences. Zinc fingers form parts of some transcription factors, which are proteins that recognize DNA base sequences during the replication and transcription of DNA. Each of the nine or ten Zn2+ ions in a zinc finger helps maintain the finger's structure by coordinately binding to four amino acids in the transcription factor. The transcription factor wraps around the DNA helix and uses its fingers to accurately bind to the DNA sequence. Copper, like all other transitional metals, forms coordination complexes. In aqueous solution, copper(II) exists as [Cu(H2O)6]2+. This complex exhibits the fastest water exchange rate (speed of water ligands attaching and detaching) for any transition metal aquo complex. Adding aqueous sodium hydroxide causes the precipitation of light blue solid copper(II) hydroxide. A simplified equation is: Cu2+ + 2 OH− → Cu(OH)2 96 Aqueous ammonia results in the same precipitate. Upon adding excess ammonia, the precipitate dissolves, forming tetraamminecopper(II): Cu(H2O)4(OH)2 + 4 NH3 → [Cu(H2O)2(NH3)4]2+ + 2 H2O + 2 OH− Many other oxyanions form complexes; these include copper(II) acetate, copper(II) nitrate, and copper(II) carbonate. Copper(II) sulfate forms a blue crystalline pentahydrate, which is the most familiar copper compound in the laboratory. It is used in a fungicide called the Bordeaux mixture. Polyols, compounds containing more than one alcohol functional group, generally interact with cupric salts. For example, copper salts are used to test for reducing sugars. Specifically, using Benedict's reagent and Fehling's solution the presence of the sugar is signaled by a color change from blue Cu(II) to reddish copper(I) oxide. Schweizer's reagent and related complexes with ethylenediamine and other amines dissolve cellulose.[34] Amino acids form very stable chelate complexes with copper(II). Many wet-chemical tests for copper ions exist, one involving potassium ferrocyanide, which gives a brown precipitate with copper(II) salts. Copper proteins are proteins that contain one or more copper ions as prosthetic groups. The metal centers in the copper proteins can be classified into several types: Type I copper centers (T1Cu) (Figure 3) are characterized by a single copper atom coordinated by two 97 histidine residues and a cysteine residue in a trigonal planar structure, and a variable axial ligand. Figure 3. Structure of poplar plastocyanin In class I T1Cu proteins (e.g. amicyanin, plastocyanin and pseudoazurin) the axial ligand is the sulfur of methionine, whereas aminoacids other than methionine (e.g. glutamine) give rise to class II T1Cu copper proteins. Azurins contain the third type of T1Cu centres: besides a methionine in one axial position, they contain a second axial ligand (a carbonyl group of a glycine residue). T1Cu-containing proteins are usually called "cupredoxins", and show similar three-dimensional structures, relatively high reduction potentials (> 250 mV), and strong absorption near 600 nm (due to S→Cu charge transfer), which usually gives rise to a blue colour. Cupredoxins are therefore often called "blue copper proteins". This may be misleading, since some T1Cu centres also absorb around 460 nm and are therefore green. When studied by EPR spectroscopy, T1Cu centres show small hyperfine splittings in the parallel region of 98 the spectrum (compared to common copper coordination compounds). Type II copper centres (T2Cu) exhibit a square planar coordination by N or N/O ligands. They exhibit an axial EPR spectrum with copper hyperfine splitting in the parallel region similar to that observed in regular copper coordination compounds. Since no sulfur ligation is present, the optical spectra of these centres lack distinctive features. T2Cu centres occur in enzymes, where they assist in oxidations or oxygenations.[2] Type III copper centres (T3Cu) consist of a pair of copper centres, each coordinated by three histidine residues. These proteins exhibit no EPR signal due to strong antiferromagnetic coupling (i.e. spin pairing) between the two S = 1/2 metal ions due to their covalent overlap with a bridging ligand. These centres are present in some oxidases and oxygentransporting proteins (e.g. hemocyanin and tyrosinase). Binuclear Copper A centres (CuA) (Figure 4) are found in cytochrome c oxidase and nitrous-oxide reductase (EC 1.7.99.6). The two copper atoms are coordinated by two histidines, one methionine, a protein backbone carbonyl oxygen, and two bridging cysteine residues. 99 Figure 4. Crystal structure of hexameric haemocyanin Copper B centres (CuB) are found in cytochrome c oxidase. The copper atom is coordinated by three histidines in trigonal pyramidal geometry. Tetranuclear Copper Z centre (CuZ) is found in nitrousoxide reductase. The four copper atoms are coordinated by seven histidine residues and bridged by a sulfur atom. Copper proteins have diverse roles in biological electron transport and oxygen transportation, processes that exploit the easy interconversion of Cu(I) and Cu(II). The biological role for copper commenced with the appearance of oxygen in earth's atmosphere. The protein hemocyanin is the oxygen carrier in most mollusks and some arthropods such as the horseshoe crab (Limulus polyphemus). Because hemocyanin is blue, these 100 organisms have blue blood, not the red blood found in organisms that rely on hemoglobin for this purpose. Structurally related to hemocyanin are the laccases and tyrosinases. Instead of reversibly binding oxygen, these proteins hydroxylate substrates, illustrated by their role in the formation of lacquers. Copper is also a component of other proteins associated with the processing of oxygen. In cytochrome c oxidase, which is required for aerobic respiration, copper and iron cooperate in the reduction of oxygen. Copper is also found in many superoxide dismutases, proteins that catalyze the decomposition of superoxides, by converting it (by disproportionation) to oxygen and hydrogen peroxide: 2 HO2 → H2O2 + O2. Several copper proteins, such as the "blue copper proteins", do not interact directly with substrates, hence they are not enzymes. These proteins relay electrons by the process called electron transfer Vanadium is often taken up as vanadate, in a pathway parallel to phosphate. However, its oxidation state within organisms seems to be highly variable.Unusually high concentrations of vanadium occur in certain ascidians (the specific transport behavior of which will be dealt with later). The workers who first characterized the vanadium-containing compound of the tunicate, Ascidia nigra, coined the name tunichrome. The characterization of the compound as a dicatecholate has been reported. Quite a different chemical environment is found in the vanadium-containing material isolated from the mushroom Amanita muscaria. Bayer and Kneifel, who named and first described amavadine, also suggested the structure shown in Figure 5. Recently the preparation, proof of ligand structure, and 101 (by implication) proof of the complex structure shown in Figure 5 have been established. Figure 5. A structure proposed for amavadine. The role of chromium in biology remains even more mysterious. In human beings the isolation of "glucose tolerance factor" and the discovery that it contains chromium goes back some time. This has been well reviewed by Mertz, who has played a major role in discovering what is known about this elusive and apparently quite labile compound. It is well established that chromium is taken up as chromic ion, predominantly via foodstuffs, such as unrefined sugar, which presumably contain complexes of chromium, perhaps involving sugar hydroxyl groups. Studies using radioactively labeled chromium have shown that, although inorganic salts of chromium are relatively unavailable to mammals, brewer's yeast can convert the chromium into a usable form. Although chromium is essential in milligram amounts for human beings as the trivalent ion, as chromate it is quite toxic and a recognized carcinogen. The uptake-reduction model for 102 chromate carcinogenicity as suggested Wetterhahn is shown in Figure 6. by Connett and Figure 6. The uptake-reduction model for chromate carcinogenicity. Possible sites for reduction of chromate include the cytoplasm, endoplasmic reticulum, mitochondria, and the nucleus Chromate is mutagenic in bacterial and mammalian cell systems, and it has been hypothesized that the difference between chromium in the +6 and +3 oxidation states is explained by the' 'uptake-reduction" model. Chromium(III), like the ferric ion discussed above, is readily hydrolyzed at neutral pH and 103 extremely insoluble. Unlike Fe3+ , it undergoes extremely slow ligand exchange. For both reasons, transport of chromium(III) into cells can be expected to be extremely slow unless it is present as specific complexes; for example, chromium(III) transport into bacterial cells has been reported to be rapid when iron is replaced by chromium in the siderophore iron-uptake mediators. However, chromate readily crosses cell membranes and enters cells, much as sulfate does. Because of its high oxidizing power, chromate can undergo reduction inside organelles to give chromium(III), which binds to small molecules, protein, and DNA, damaging these cellular components. In marked contrast to its congener, molybdenum is very different from chromium in both its role in biology and its transport behavior, again because of fundamental differences in oxidation and coordination chemistry properties. In contrast to chromium, the higher oxidation states of molybdenum dominate its chemistry, and molybdate is a relatively poor oxidant. Molybdenum is an essential element in many enzymes, including xanthine oxidase, aldehyde reductase, and nitrate reductase. The range of oxidation states and coordination geometries of molybdenum makes its bioinorganic chemistry particularly interesting and challenging. Common oxidation states of cobalt include +2 and +3, although compounds with oxidation states ranging from −3 to +4 are also known. A common oxidation state for simple compounds is +2. Cobalt(II) salts form the red-pink [Co(H2O)6]2+ complex in aqueous solution. Addition of chloride gives the intensely blue [CoCl4]2−.[ The principles of electronegativity and hardness–softness of a series of ligands can be used to explain the usual oxidation state of the cobalt. For example Co+3 complexes tend to have ammine ligands. As phosphorus is softer than nitrogen, phosphine 104 ligands tend to feature the softer Co2+ and Co+, an example being tris(triphenylphosphine)cobalt(I) chloride ((P(C6H5)3)3CoCl). The more electronegative (and harder) oxide and fluoride can stabilize Co4+ and Co5+ derivatives, e.g., caesium hexafluorocobaltate (Cs2CoF6) and potassium percobaltate (K3CoO4).[ Figure 7. Structure of Co complexes with biomolecules 105 In the living bodies, cobalt is found in coenzyme B12 (Figure 7), which is essential for the transfer of alkyl groups from one molecule to another in biological systems, as well as the reduction of the ribose ring in ribonucleotides that make up RNA to the deoxyribose ring in deoxyribonucleotides that make up DNA. The cobalamin-based proteins use corrin to hold the cobalt. Coenzyme B12 features a reactive C-Co bond, which participates in its reactions. In humans, B12 exists with two types of alkyl ligand: methyl and adenosyl. MeB12 promotes methyl (CH3) group transfers. The adenosyl version of B12 catalyzes rearrangements in which a hydrogen atom is directly transferred between two adjacent atoms with concomitant exchange of the second substituent, X, which may be a carbon atom with substituents, an oxygen atom of an alcohol, or an amine. Methylmalonyl coenzyme A mutase (MUT) converts MMl-CoA to Su-CoA, an important step in the extraction of energy from proteins and fats. Although far less common than other metalloproteins (e.g. those of zinc and iron), cobaltoproteins are known aside from B12. These proteins include methionine aminopeptidase 2 an enzyme that occurs in humans and other mammals which does not use the corrin ring of B12, but binds cobalt directly. Another non-corrin cobalt enzyme is nitrile hydratase, an enzyme in bacteria that are able to metabolize nitriles. 106 CHAPTER III Structure, properties, and functions of metal complexes of proteins and nucleic acids Part 1. Metalloprotein structures Proteins are large biological molecules consisting of one or more chains of -amino acids: The structure of proteins includes: •primary structure: sequence of amino acids; •secondary structure: shapes formed within regions of the protein; •tertiary structure: shape of entire protein; •quaternary structure: structures formed by interaction of several subunit. Proteins perform a vast array of functions within living organisms, including catalyzing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific three-dimensional structure that determines its activity. 107 It is estimated that approximately half of all proteins contain a metal. In another estimate, about one quarter to one third of all proteins are proposed to require metals to carry out their functions. Amino acids are important low molecular weight ligands in biological systems as metals are bound to proteins through amino acid side chains: 108 Coordination through carboxylate has three basic modes: Characteristic affinities of some amino acids to defined oxidation states of metals, typical coordination numbers and coordination geometries are presented below: Metal centers are undersaturated (bonding of substrate), and their coordination geometries are frequently distorted. So, the possibility of extra coordination of some exogenous ligands 109 which are not integrated into the active site of the protein exists. The most common of them are the following: (i) water-derived (H2O, OH-); (ii) oxygen-derived (O2, O2-, O22-, HO2-); (iii) neutral species (CO, NO); (iv) halides and other charged species (Cl-, PO43-). Systematic of copper proteins Type I (Blue Cu-proteins): trigonal coordination geometry; ligands: 2 Cys(1-), 1 His, 1 weakly bonded Met. Function: e- transfer; Example: Plastocyanin in the e- transfer chain PSII→PSI. Type II: Tetragonal coordination geometry; ligands: His, Tyr(1-), H2O, no Cys. Function: Redox reactions; Examples: Galactoseoxidase (RCH2OH → RCHO + 2H+ e-), Cu/Zn superoxidedismutase (2O2- + 2H+ → O2 + H2O2). Type III: Contains 2 cooperating Cu centers; trigonal coordination geometry; ligands: 3 His per Cu. Intensely blue in the oxidized form (O22- →Cu2+ LMCT). Function: Transport and transfer (to a substrate) of oxygen; examples: haemocyanin (Figure 8); tyrosinase (Tyr + ½O2 + 2e- → DOPA). Figure 8. Reversible uptake and release of oxygen by haemocyanins 110 Ceruloplasmin, important for the absorption of iron, is a Cu protein containing 7 Cu centers representing types I, II and III. Nitritereductase contains type II (substrate activation) and type I Cu centres (e- transfer). Among the others, there are CuA und CuB in cytochrom-c oxidase (Figure 9). Figure 9. Organisation of the redox-active centres of cytochromec oxidase (left). Oxygen activation and reduction occurs at the dinuclear CuB⋅⋅⋅Cyt-a3 pair (see expansion to the right). 111 Iron-Sulfur Proteins. Transition-metal/sulfide sites, especially those containing iron, are present in all forms of life and are found at the active centers of a wide variety of redox and catalytic proteins. These proteins include simple soluble electron-transfer agents (the ferredoxins), membrane-bound components of electron-transfer chains, and some of the most complex metalloenzymes, such as nitrogenase, hydrogenase, and xanthine oxidase. The most important (in the sense that they are more generally used) members of this family are collated in Figure 10. (1) Rubredoxins contain one iron centre tetrahedrally coordinated to four cysteinates. (2) [2Fe,2S] ferredoxines, with two iron centers, constitute twoedge-bridged FeS4 tetrahedra. The bridging sulphur functions are inorganic sulphide S2-, the remaining ligands are cysteinates. (3) [4Fe,4S] ferredoxins have a cubane structure. The four trebly bridging functions are again sulfide, also termed labile sulfur because they can be converted to volatile H2S with acids. The mean oxidation state in the reduced form is 2.25, in the oxidized form 2.5, the redox potential is typically around -200 mV. (4) HiPIPs (High Potential Iron Proteins) are identical to the [4Fe,4S] ferredoxins in as far as the core structure is concerned. However, the mean oxidation state in the reduced form is 2.5, in the oxidized from 2.75, and the redox potential is typically around +300 mV. Along with these “classical” iron/sulfur clusters, others are know, in which one iron centre is missing ([3Fe,4S] ferredoxins), or where two [4Fe,4S] ferredoxins form double-cubanes, or where a fifth ligand (Ser or His) is coordinated to one of the iron centers. 112 Figure 10. The iron centres of the classical (and more frequently used) iron-sulphur proteins. SR = cysteinate(1-). Iron/sulfur proteins are for example included in the mitochondrial respiratory chain. The reduction of O2 to H2O takes place step by step in order to prevent damage to cellular constituents by the burst of energy liberated in a single step. The reaction cascade is termed respiratory chain, which takes place in the mitochondria, and serves the generation of energy. The process is illustrated in the Figure 11. 113 Figure 11. Reaction cascade in the mitochondrial respiratory chain (shortened). Iron sulfide proteins are also involved involved in electron transfer processes. The Figure 12 shows the ranges of redox behavior known for Fe-S centers. Clearly, the Fe-S systems can carry out low-potential processes. The rubredoxins cover the midpotential range, and the HiPIPs are active in the high-potential region. 114 Figure 12. A schematic diagram of the redox potential of the various FeS centers in comparison with other known redox centers. Part 2. Nucleic Acids. Nucleic acids are large biological molecules essential for all known forms of life. They include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Together with proteins, nucleic acids are the most important biological macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information. Nucleic acids are linear polymers (chains) of nucleotides. Each nucleotide consists of three components: a purine or pyrimidine nucleobase (sometimes termed nitrogenous base or 115 simply base), a pentose sugar, and a phosphate group. The substructure consisting of a nucleobase plus sugar is termed a nucleoside. Nucleic acid types differ in the structure of the sugar in their nucleotides - DNA contains 2'-deoxyribose while RNA contains ribose (where the only difference is the presence of a hydroxyl group). Also, the nucleobases found in the two nucleic acid types are different: adenine, cytosine, and guanine are found in both RNA and DNA, while thymine occurs in DNA and uracil occurs in RNA. The sugars and phosphates in nucleic acids are connected to each other in an alternating chain (sugar-phosphate backbone) through phosphodiester linkages.[10] In conventional nomenclature, the carbons to which the phosphate groups attach are the 3'-end and the 5'-end carbons of the sugar. This gives nucleic acids directionality, and the ends of nucleic acid molecules are referred to as 5'-end and 3'-end. The nucleobases are joined to the sugars via an N-glycosidic linkage involving a nucleobase ring nitrogen (N-1 for pyrimidines and N-9 for purines) and the 1' carbon of the pentose sugar ring. The structures of the components of two principal nucleic acids RNA and DNA are presented on the Figure 13. 116 Figure 13. Nucleic acids components and structure. 117 Coordination of Nucleic Acids with Metals. Most prevalent among covalent complexes with DNA are those involving coordination between soft metal ions and nucleophilic positions on the bases. For example, the structure of the fragment of cis-(NH3)2Pt-dGpG is presented on the Figure 14. It is evident that the platinum center coordinates to the N7 position of the guanine bases. In terms of interactions with the full polynucleotide, it is likely that the cis-diammineplatinum center, with two coordination sites available, would yield an intrastrand crosslink between neighboring guanine residues on a strand. Figure 14. Possible coordination modes of nucleic acids Base binding at the purine N7 position is, of course, not limited to soft metal ions such as Pt(II), Pd(II), and Ru(II). 118 Coordination at these sites has been evident also with first-row transition-metal ions such as Cu(II) and Zn(II). Transition-metal ions with decreasing softness are capable of coordinating also to the phosphate oxygen atoms. The ionic versus covalent character of these complexes clearly depends on the metal ions involved. The preference for phosphate over base association was found to decrease in the order Mg(II) > Co(II) >Ni(II) >Mn(II) >Zn(II) >Cd(II) >Cu(II). This series arises from examination of DNA helix-melting temperatures, since base interactions in general should destabilize the helical form [except where interstrand crosslinking occurs, as may happen with Ag(I)], whereas phosphate coordination and neutralization would increase the helix stability and hence the melting temperature. Also of interest, but less common, are covalent interactions with the sugar moiety. Although the pentose ring in general provides a poor ligand for metal ions, osmate esters can form quite easily across the C2'-C3' positions in the ribose rings. This particular interaction has been suggested as a basis for heavy metal staining of RNA. In fact, OsO4 is not restricted in its reactivity with the sugar positions. Intercalation and hydrogen bonding. Important interactions of metal complexes with polynucleotides are not restricted to those involving direct coordination of the metal center to the polymer. Instead, an abundance of highly selective interactions arise from an ensemble of weaker noncovalent interactions between the ligands of coordinatively saturated metal complexes and the nucleic acid. Two primary examples of noncovalent association are given by metallointercalation and hydrogen-bonding interactions of coordinated ligands (Figure 15). Planar aromatic heterocyclic ligands such as phenanthroline and terpyridine can stack in between the DNA base pairs, stabilized through dipole-dipole 119 interactions. Here, depending on the complex and its extent of overlap with the base pairs, the free energy of stabilization can vary from ~2 to 10 kcal. Nonintercalative hydrophobic interactions of coordinated ligands in the DNA grooves also can occur, as we will see. Hydrogenbonding interactions of coordinated ligands with the polynucleotide are quite common, and arise in particular with the phosphate oxygen atoms on the backbone. With cobalt hexaammine, for example, hydrogen bonding to an oligonucleotide occurs between the ammine hydrogens and both phosphate oxygen atoms and purine bases. A mix of covalent and noncovalent interactions is also possible. With cisdiammineplatinum(II) coordinated to the guanine N7 position, the ammine ligands are well-poised for hydrogen-bonding interactions with the phosphate backbone. The steric constraints on the molecule must be, however, considered. 120 (a) (b) Figure 15. Examples of intercalation (a) and hydrogen bonding (b) in the processes of interaction of metal ions with nucleic acids 121 Fundamental Reactions with Nucleic Acids Complexes. The reactions of transition-metal complexes with polynucleotides generally fall into two categories: (i) those involving a redox reaction of the metal complex that mediates oxidation of the nucleic acid; and (ii) those involving coordination of the metal center to the sugar-phosphate backbone so as to mediate hydrolysis of the polymer. 1. Redox chemistry. The simplest redox reaction with polynucleotides one might consider as an illustration is the Fenton reaction, which indirectly promotes DNA strand scission through radical reactions on the sugar ring. The reaction with Fe(EDTA)2 is shown in Figure 16. As do other redox-active divalent metal ions, ferrous ion, in the presence of hydrogen peroxide, generates hydroxyl radicals, and in the presence of a reductant such as mercaptoethanol, the hydroxyl radical production can be made catalytic. Although ferrous ion itself does not appear to interact appreciably with a nucleic acid, especially when chelated in an anionic EDTA complex and repelled by the nucleic-acid polyanion, the hydroxyl radicals, produced in appreciable quantities catalytically, attack different sites on the sugar ring, indirectly yielding scission of the sugar-phosphate backbone. 122 Figure 16. Scheme of a redox-reaction with Fe(EDTA)2 Metal ions can also be used to generate other oxidizing intermediates in aerated aqueous solution, such as superoxide ion and singlet oxygen. 2. Hydrolytic chemistry. Hydrolysis reactions of nucleic acids mediated by metal ions are important elements in natural enzymatic reactions; 123 chemists would like to exploit them in the design of artificial restriction endonucleases. Hydrolysis reactions of the phosphodiester linkage of polynucleotides appear preferable to redox-mediated cleavage reactions, since in the hydrolytic reaction all information is preserved. In redox cleavage by sugar oxidation, for example, both a sugar fragment and free nucleic-acid base are released from the polymer, and, in contrast to hydrolytic chemistry, the direct relegation of the fragments becomes practically impossible. Metal ions can be effective in promoting hydrolysis of the phosphodiester, since they can function as Lewis acids, polarizing the phosphorus-oxygen bond to facilitate bond breakage, and can also deliver the coordinated nucleophile to form the pentacoordinate phosphate intermediate. Last, it must be mentioned that metal coordination to the purine N7 position can also indirectly promote strand cleavage, although not through direct hydrolytic reaction on the sugarphosphate backbone. Metal ions such as Pd2+ and Cu2+, through coordination at N7, promote depurination. The depurinated site then becomes easily susceptible to hydrolysis upon treatment with mild base: 124 Hydrolysis reactions catalyzed by metal ions and complexes: (a) Illustration of a phosphate ester hydrolysis in a binuclear model complex catalyzed by coordinated cobaltic ions, with one metal ion functioning as a Lewis acid and the other functioning to deliver the coordinated hydroxide. (b) Ru(DIPh)Macro, a metal complex constructed to contain a central DNA-binding domain (Ru(DIP)32+) with two tethered amine arms to chelate additional metal ions (Zn2+) to deliver to the sugar-phosphate backbone and promote hydrolytic strand cleavage. (c) RNA site-specificially hydrolyzed by lead ion. 125 CHAPTER IV Transition Metal Storage, Transport and Biomineralization Living organisms store and transport transition metals both to provide appropriate concentrations of them for use in metalloproteins or cofactors and to protect themselves against the toxic effects of metal excesses; metalloproteins and metal cofactors are found in plants, animals, and microorganisms. In multicellular organisms, composed of a variety of specialized cell types, the storage of transition metals and the synthesis of the transporter molecules are not carried out by all types of cells, but rather by specific cells that specialize in these tasks. The form of the metals is always ionic, but the oxidation state can vary, depending on biological needs. Transition metals for which biological storage and transport are significant are, in order of decreasing abundance in living organisms: iron, zinc, copper, molybdenum, cobalt, chromium, vanadium, and nickel. Iron is the most common transition metal in biology. 6,7 Its use has created a dependence that has survived the appearance of dioxygen in the atmosphere ca. 2.5 billion years ago, and the concomitant conversion of ferrous ion to ferric ion and insoluble rust. The average amount of iron in the human body (70 kg) is ca. 5 g; iron is thus the most abundant transition metal in our organism. About 70% of this amount is used for oxygen transport and storage (haemoglobin, myoglobin), almost 30% are stored in ferritins (iron storage proteins), and about 1% is bound to the transport protein transferrin and to various irondependent enzymes: 126 Haeme-type (e.g. cytochromes, haemoglobin) Iron-sulfur proteins (e.g. ferridoxines, Rieske proteins) Two iron centers (e.g. ribonucleotidereductase) Three properties of iron can account for its extensive use in terrestrial biological reactions: (a) facile redox reactions of iron ions; (b) an extensive repertoire of redox potentials available by ligand substitution or modification; (c) abundance and availability under conditions apparently extant when terrestrial life began. The processes and reactions in which iron participates are crucial to the survival of terrestrial organisms, and include ribonucleotide reduction (DNA synthesis), energy production (respiration), energy conversion (photosynthesis), nitrogen reduction, oxygen transport (respiration, muscle contraction), and oxygenation (e.g., steroid synthesis, solubilization and detoxification of aromatic compounds). On the other hand, Fe(III) has a low solubility under physiological conditions ( 10-18 M), requiring living organisms to adapt more efficient iron storage/transport/usage mechanisms. 127 The Storage of Iron Iron is stored mainly in the ferritins. Ferritin is a globular protein complex consisting of 24 protein subunits (Figure 17, 18) and is the primary intracellular iron-storage protein in both prokaryotes and eukaryotes, keeping iron in a soluble and nontoxic form. Ferritin that is not combined with iron is called apoferritin. Figure 17. Structure of the murine ferritin complex Figure 18. Crystallographic structure of mitochondrial ferritin 128 Ferritin is found in animals, plants, and even in bacteria; the role of the stored iron varies, and includes intracellular use for Fe-proteins or mineralization, long-term iron storage for other cells, and detoxification of excess iron. Iron regulates the synthesis of ferritin, with large amounts of ferritin associated with iron excess, small or undetectable amounts associated with iron deficiency. The structure of ferritin is the most complete paradigm for bioinorganic chemistry because of three features: the protein coat, the iron-protein interface, and the iron core. Protein Coat consists of twenty-four peptide chains (with about 175 amino acids each), folded into ellipsoids, pack to form the protein coat, which is a hollow sphere about 100 Å in diameter; the organic surface is about 10 A thick (Figure 17). Channels which occur in the protein coat at the trimer interfaces may be involved in the movement of iron in and out of the protein. Since the protein coat is stable with or without iron, the center of the hollow sphere may be filled with solvent, with Fe2O3 nH2O, or, more commonly, with both small aggregates of iron and solvent. Very similar amino-acid sequences are found in ferritin from animals and plants. Sorting out which amino acids are needed to form the shape of the protein coat and the ligands for iron core formation requires the continued dedication of bioinorganic chemists; identification of tyrosine as an Fe(III)ligand adds a new perspective. Iron-Protein Interface Formation of the iron core appears to be initiated at an Fe-protein interface where Fe(II)-O-Fe(Ill) dimers and small clusters of Fe(III) atoms have been detected attached to the protein and bridged to each other by oxo/hydroxo bridges. Iron Core Only a small fraction of the iron atoms in ferritin bind directly to the protein. The core contains the bulk of the iron in a polynuclear aggregate with properties similar to 129 ferrihydrite, i.e. hydrous ferric oxide [Fe2O3(H2O)n] with various amounts of phosphate. The overall composition of the iron nucleus is 8FeO(OH)·FeO(H2PO4). Channels of threefold symmetry and a width of 10 Å allow for an exchange of Fe3+ between the interior and exterior. As many as 4,500 iron atoms can be reversibly stored inside the protein coat in a complex that is soluble; iron concentrations equivalent to 0.25 M [about 10 16-fold more concentrated than Fe(III) ions] can be easily achieved in vitro. Magnetite (Fe3O4) is another form of biological iron derived, apparently, from the iron in ferritin. Magnetite plays a role in the behavior of magnetic bacteria, bees, and homing pigeons. Ferritins – like many other proteins – exhibit high symmetry. High symmetry (also found with higher organised forms of life such as viruses, bacteria and even proteins in plants and animals) makes less reactive – as a consequence of minimised overall polarity – and thus has “a protective function”. For some basic considerations on symmetry, also of relevance in the context of the electronic configuration of metal ions in coordination compounds The Uptake and Transport of Iron The extremely low solubility of the stored Fe(OH)3 [solubility product L = 2·10-39, solubility (pH 7) l =10-18 mol·l-1], and thus the unavailability of iron in aqueous media under oxic conditions, has forced many groups of organisms to develop suitable systems for the mobilisation of iron. These systems, socalled siderophores excreted by the organisms, are multidentate anionic ligands which form extremely stable complexes with Fe3+ (complex formation constants up to 1050 M-1). The functional groups of these ligands are, in many cases, catecholates (o130 hydroxyquinolates), as in the case of enterobactin, or hydroxamates (ferrioxamines and ferriochromes) (Figure 19). The complexes are more or less globular, with the outer sphere furnished with hydrophilic groups (amide and ester groups), allowing for the water solubility and easy transport in the aquatic medium. Internalization of the iron-loaded siderophore by the organism typically takes place by endocytosis; the cytosolic remobilisation of the iron either by reduction of Fe3+ to Fe2+ and recycling of the siderophore, or by oxidative destruction of the siderophore. (a) (b) Figure 19. Structures of enterobactin (a) and ferrioxamine E (b) 131 Iron, when taken up with the food and processed in the mouth (chewing, admixture of saliva) is mostly present in its ferric (Fe3+) form and thus gets into the gastro-intestinal tract as Fe3+. In case of an intact milieu in the small intestines, ferric iron is reduced to its ferrous form (Fe2+). Only in this oxidation state can iron be absorbed by the epithelium cells of the mucosa. For transfer to the blood serum, reoxidation to Fe3+ is necessary. The oxidation Fe2+ → Fe3+ + e- in the mucosa is catalyzed by a copper enzyme (ceruloplasmin, containing 7 copper centres: Cu+ → Cu2+). The Fe3+ ions are then taken up by apotransferrin (H2Tf); simultaneously, carbonate is coordinated to iron. Fe3+-Tf is the transport form for iron. The iron-loaded transferrin, (Figure 20) delivers iron to sites of potential use (e.g. incorporation into protoporphyrin IX and generation of haemoglobin), or stored in iron storage proteins (ferritins). The delivery of iron affords reduction from the ferric to the ferrous state; a reductant employed here is ascorbate (vitamin C): uptake: H2Tf + Fe3+ + HCO3- → [(Tf)FeIII(CO3)]- + 3H+ release: [(Tf)FeIII(CO3)]- + e- + 3H+ → H2Tf + HCO3- + Fe2+ 132 Figure 20. The FeIII – carbonate – transferring complex. Biomineralization of Iron Biomineralization is the process by which living organisms produce minerals, often to harden or stiffen existing tissues. Such tissues are called mineralized tissues. Biologically formed iron ore is by far the most common and thoroughly studied process of biomineralization There are five major variations on iron biomineralization that incorporates both BIM and BCM: Hydroxides and oxides, phosphates, silicates, sulfates and sulfides. Hydroxides and oxides. Iron oxides, as one of the beststudied classes of biominerals containing transition metals. Three different forms of biological iron oxides appear to have distinct 133 relationships to the proteins, lipids, or carbohydrates associated with their formation and with the degree of crystallinity. Magnetite, on the one hand, often forms almost perfect crystals inside lipid vesicles of magneto-bacteria. Ferrihydrite, on the other hand, exists as large single crystals, or collections of small crystals, inside the protein coat of ferritin; however, iron oxides in some ferritins that have large amounts of phosphate are very disordered. Finally, goethite [-FeO(OH)] and lepidocrocite [-FeO(OH)] form as small single crystals in a complex matrix of carbohydrate and protein in the teeth of some shellfish (limpets and chitons); magnetite is also found in the lepidocrocitecontaining teeth. The differences in the iron-oxide structures reflect differences in some or all of the following conditions during formation of the mineral: nature of co-precipitating ions, organic substrates or organic boundaries, surface defects, inhibitors, pH, and temperature. Magnetite can form in both lipid and protein/carbohydrate environments, and can sometimes be derived from amorphous or semicrystalline ferrihydrite-like material (ferritin). However, the precise relationship between the structure of the organic phase and that of the inorganic phase has yet to be discovered. Synthetic iron complexes have provided models for two stages of ferritin iron storage and biomineralization: (1) the early stages, when small numbers of clustered iron atoms are bound to the ferritin protein coat, and (2) the final stages, where the bulk iron is a mineral with relatively few contacts to the protein coat. In addition, models have begun to be examined for the microenvironment inside the protein coat (Figure 21). 134 Figure 21. The structure of a model for a possible intermediate in the formation of the ferritin iron core. The complex consists of 11 Fe(III) atoms with internal oxo-bridges and a coat of benzoate ligands; the Fe atoms define a twisted, pentacapped trigonal prism. Sulfides. Following ores that are the result of oxidization, the sulfides, formed in anoxic environments are probably the most important ores for mining. Pyrite is one of the most common iron sulfides and has been connected in some cases to bacterial activities. Sulfides are the end-products of bacterial metabolism of the energy rich sulfates. The sulfide generated by the bacteria reacts with iron oxides or hydroxides and form 135 monosulfide phases as FeS (and mackinawite (Fe,Ni)1-2S) but also elemental sulfur. The latter then becomes the oxidant required to convert FeS to pyrite FeS2. Sulfates. As a result of oxidization in acidic conditions of ferrihydrates and also iron sulfides bacteria can produce ferric hydroxysulfates. The exact process of this in detail on a bacterial level is still a matter of debate. Phosphates. Phosphates are a common source of energy for bacteria and occur in abundance in, for example anoxic water environments. Phosphate is often dissolved from underlaying apatite (Ca5(PO4)3(F,Cl,OH)) rich rocks or a product from the decay of organic material. The formation of iron phosphate minerals through microbes such as strengite (FePO42H2O), which is a hydrated iron phosphate created by organic acids, has been directly observed when analyzing biofilms growing on phosphorite sediments. Associations with simple iron oxides (FeO) and goethite has also been found in phosphorite sediments from Paleozoic to Cenozoic times. These biofilms, or lichens, which are symbiotical concentrations of bacteria and fungi concentrate and immobilize metals from the phosphates. Phosphorite sediments can sometime form banded layers of hundred of meters in thickness with thinner layers of phosphorous iron ores. Silicates. Bacteria can also form iron rich silicates, primarily found in acidic hot spring sediments. Examination of such sediments has revealed bacterial cells completely encrusted in these minerals. They are presumed to be formed as a result of iron binding to anionic cells and where dissolved silica in the spring then was subsequently added to the growing minerals in 136 the process. Another process in which silicates form has been observed in biofilms in clays in freshwater environments. Here (Fe, Al)-silicates similar to chamonite ((Fe5Al)(Si3Al)10(OH)8) and kaolinite (Al4(Si4O10)(OH)4), but with a poor crystallinity was found. These are therefore thought to have formed as a result of reaction between the silica, metals and possibly metals within the cell since it is well known that fresh water bacteria can bind and immobilize metals. The process of forming a silicate is then completed through diagenesis. Conclusion Transition play key roles in such biological processes. Among them, Fe predominates in terrestial abundance; since Fe is involved in a vast number of biologically important reactions, its storage and transport have been studied extensively. Two types of Fe carriers are known: specific proteins and low-molecularweight complexes. In higher animals, the transport protein transferrin binds two Fe atoms with high affinity; in microorganisms, iron is transported into cells complexed with catecholates or hydroxamates called siderophores; and in plants, small molecules such as citrate, and possibly plant siderophores, carry Fe. Iron complexes enter cells through complicated paths involving specific membrane sites (receptor proteins). A problem yet to be solved is the form of iron transported in the cell after release from transferrin or siderophores but before incorporation into Fe-proteins. The problems of storage, transport and biomineralization of other transitional metals, such as Cu, Mo, Cr, Co, Mn, V and Zn are proposed as research topics for the students and are not discussed here. 137 CHAPTER V Biological dioxygen transport system The study of oxygen transfer in animals and plants represents a large and diverse field of research, covering not only different levels of organisation ranging from the whole organism through to subcellular organelles, and different mechanisms such as convection and simple or facilitated diffusion, but also, of necessity, invoking diverse methodological approaches. Most organisms require molecular oxygen in order to survive. The dioxygen isused in a host of biochemical transformations, although most is consumed in the reaction O2 + 4H+ + 4e- 2H2O that is the terminal (or primary) step of oxidative phosphorylation. An elegant three-component system has evolved to transport dioxygen from regions of high abundance (water and air) to regions of relatively low abundance and high demand (the interior cells of the organism). This process is illustrated in Figure 22. 138 Figure 22. Oxygen sequestration and transport in the generalized organism. The central component is a dioxygen-carrier protein where the dioxygen-binding site, that is, the so-called "active site," is a complex either of iron or of copper. The second component of the dioxygen-transport system facilitates the sequestration of dioxygen by the dioxygen-carrier protein. Specialized organs, such as lungs in air-breathing creatures or gills in fish, offer a very large surface area to the outside environment in order to facilitate diffusion. 139 The third component is the delivery system. The oxygen carrier is dissolved or suspended in a fluid, called blood plasma or hemolymph, that is pumped throughout the animal by another specialized organ, the heart, through a network of tubes, the blood vessels. Structure and characteristics of dioxygencarrier proteins Hemoglobins are the most widely distributed family of dioxygen carriers. Their active site consists of an iron porphyrin (heme) group embedded in the protein. Hemoglobin is a globular protein (i.e., folded into a compact, nearly spherical shape) and consists of four subunits, as shown in Figure 23. Each protein subunit is an individual molecule that joins to its neighboring subunits through intermolecular interactions. Figure 23. The molecular model of hemoglobin. 140 Each subunit contains a heme group, which is displayed using the ball-and-stick representation in Figure 23. Each heme group contains an iron atom that is able to bind to one oxygen (O2) molecule. Therefore, each hemoglobin protein can bind four oxygen molecules. In the body, the iron in the heme is coordinated to the four nitrogen atoms of the porphyrin and also to a nitrogen atom from a histidine residue (one of the amino-acid residues in hemoglobin) of the hemoglobin protein (Figure 24). The sixth position (coordination site) around the iron of the heme is occupied by O2 when the hemoglobin protein is oxygenated. Figure 24. The coordination mode of iron in a deoxygenated hemoglobin. 141 The heme group is nonplanar when it is not bound to oxygen; the iron atom is pulled out of the plane of the porphyrin, toward the histidine residue to which it is attached. This nonplanar configuration is characteristic of the deoxygenated heme group, and is commonly referred to as a "domed" shape. The valence electrons in the atoms surrounding iron in the heme group and the valence electrons in the histidine residue form "clouds" of electron density. (Electron density refers to the probability of finding an electron in a region of space.) Because electrons repel one another, the regions occupied by the valence electrons in the heme group and the histidine residue are pushed apart. Hence, the porphyrin adopts the domed (nonplanar) configuration and the Fe is out of the plane of the porphyrin ring (Figure 25, left). However, when the Fe in the heme group binds to an oxygen molecule, the porphyrin ring adopts a planar configuration and hence the Fe lies in the plane of the porphyrin ring (Figure 25, right). Figure 25. Electron-density clouds of the deoxygenated heme group (pink) and the attached histidine residue (light blue) in deoxygenated (left) and oxygenated (right) hemoglobin 142 The shape change in the heme group has important implications for the rest of the hemoglobin protein, as well. When the iron atom moves into the porphyrin plane upon oxygenation, the histidine residue to which the iron atom is attached is drawn closer to the heme group. This movement of the histidine residue then shifts the position of other amino acids that are near the histidine (Figure 26). When the amino acids in a protein are shifted in this manner (by the oxygenation of one of the heme groups in the protein), the structure of the interfaces between the four subunits is altered. Hence, when a single heme group in the hemoglobin protein becomes oxygenated, the whole protein changes its shape. In the new shape, it is easier for the other three heme groups to become oxygenated. Thus, the binding of one molecule of O2 to hemoglobin enhances the ability of hemoglobin to bind more O2 molecules. This property of hemoglobin is known as "cooperative binding." Hemoglobin exists in two forms, a taut (tense) form (T) and a relaxed form (R). Various factors such as low pH, high CO2 and high 2,3-Bisphosphoglyceric acid (2,3-Bisphosphoglycerate or 2,3-BPG) at the level of the tissues favor the taut form, which has low oxygen affinity and releases oxygen in the tissues. Conversely, a high pH, low CO2, or low 2,3 BPG favors the relaxed form which can better bind oxygen. The partial pressure of the system also affects O2 affinity where, at high partial pressures of oxygen, the tense state is favored. Inversely, at low partial pressures, the relaxed state is favored. Additionally, the binding of oxygen to the Iron-II heme pulls the iron into the plane of the porphryn ring, causing a slight conformational shift. The shift encourages other similar ligands to bind to the three remaining hemes within hemoglobin (thus, oxygen binding is cooperative). 143 Figure 26. Structures of deoxygenated (left) and oxygenated (right) forms of hemoglobin The structure of hemoglobins varies across species. Hemoglobin occurs in all kingdoms of organisms, but not in all organisms. Primitive species such as bacteria, protozoa, algae, and plants often have single-globin hemoglobins. Many nematode worms, molluscs, and crustaceans contain very large multisubunit molecules, much larger than those in vertebrates. In particular, chimeric hemoglobins found in fungi and giant annelids may contain both globin and other types of proteins. Among the other oxygen-binding proteins, we can mention the following: 1. Myoglobin (Figure 27) is found in the muscle tissue of many vertebrates, including humans, it gives muscle tissue a distinct red or dark gray color. It is very similar to hemoglobin in structure and sequence, but is not a tetramer; instead, it is a monomer that lacks cooperative 144 binding. It is used to store oxygen rather than transport it. Figure 27. Example of molecular structure of myoglobin and magnified view. 2. Hemocyanin (Figure 28) is the second most common oxygen-transporting protein. It is found in the blood of many arthropods and molluscs. Figure 28. Deoxygenated (left) and oxygenated (right) hemocyanin. 145 Oxygenation causes a color change between the colorless Cu(I) deoxygenated form and the blue Cu(II) oxygenated form. Hemocyanins carry oxygen in the hemolymph of most molluscs, and some arthropods. They are second only to hemoglobin in frequency of use as an oxygen transport molecule. Unlike the hemoglobin in red blood cells found in vertebrates, hemocyanins are not bound to blood cells but are instead suspended directly in the hemolymph. 3. Hemerythrin (Figure 29) is an oligomeric protein responsible for oxygen transport in the marine invertebrate phyla of sipunculids, priapulids, brachiopods, and in a single annelid worm, magelona. Figure 29. Active sites of hemerythrin before and after oxygenation. Hemerythrin does not, as the name might suggest, contain a heme fragment and represents a non-heme protein. 146 The mechanism of dioxygen binding is unusual. Most O2 carriers operate via formation of Dioxygen complexes, but hemerythrin holds the O2 as a hydroperoxide. The site that binds O2 consists of a pair of iron centres. The iron atoms are bound to the protein through the carboxylate side chains of a glutamate and aspartates as well as through five histidine residues. Hemerythrin and myohemerythrin are often described according to oxidation and ligation states of the iron centre: Fe2+—OH—Fe2+ Fe2+—OH—Fe3+ Fe3+—O—Fe3+—OOHFe3+—OH—Fe3+—R (any other ligand) deoxy (reduced) semi-met oxy (oxidized) met (oxidized) Deoxyhemerythrin contains two high-spin ferrous ions bridged by hydroxyl group (A). One iron is hexacoordinate and another is pentacoordinate. A hydroxyl group serves as a bridging ligand but also functions as a proton donor to the O2 substrate. This proton-transfer result in the formation of a single oxygen atom (μ-oxo) bridge in oxy- and methemerythrin. O2 binds to the pentacoordinate Fe2+ centre at the vacant coordination site (B). Then electrons are transferred from the ferrous ions to generate the binuclear ferric (Fe3+, Fe3+) centre 147 4. with bound peroxide (C). It appears pink/violet when oxygenated, clear when not. Chlorocruorin is present in the blood plasma of many annelids. It carries an abnormal heme group structure (Figure 30). This enormous macromolecule is typically found free floating in the plasma, and not contained within red blood cells. Figure 30. Structure of the active center of chlorocruorin 5. 148 The affinity of chlorocruorin for oxygen is weaker than that of most haemoglobins. It appears green when deoxygenated and red when oxygenated. Vanabins are also known as vanadium chromagens, they are found in the blood of sea squirts. There were once hypothesized to use the rare metal vanadium as an oxygen binding 6. prosthetic group. However, although they do contain vanadium by preference, they apparently bind little oxygen, and thus have some other function, which has not been elucidated. Erythrocruorin is found in many annelids, including earthworms, it is a giant free-floating blood protein containing many dozens – possibly hundreds – of iron- and heme-bearing protein subunits bound together into a single protein complex with a molecular mass greater than 3.5 million daltons. Requirements for Effective Oxygen Carriers In order for dioxygen transport to be more efficient than simple diffusion through cell membranes and fluids, it is not sufficient that a metalloprotein merely binds dioxygen. Not only is there an optimal affinity of the carrier for dioxygen, but also, and more importantly, the carrier must bind and release dioxygen at a rapid rate. These thermodynamic and kinetic aspects are illustrated in Figure 31 for the process where M is an oxygen carrier, for example hemocyanin. Thermodynamic or equilibrium aspects are summarized by G in Figure 31. 149 Figure 31. Schematic diagram of energy changes in dioxygen binding. As it is illustrated, G is negative, and thus the forward reaction, dioxygen binding, is spontaneous. The equilibrium constant (K) is given by where a is activity of species or in terms of concrntrations and partial pressure of oxygen: 150 It is very convenient to express the affinity as the partial pressure of dioxygen required for half-saturation of the species M, Pl/2(O2). Under such conditions, [M] = [MO2], one obtains The dioxygen affinity is composed of enthalpic (H) and entropic (S) components, with Within a family of oxygen carriers the values of ASo and Mfo are usually similar. Large deviations (such as a change of sign) are therefore indicative of a change in the nature of the oxygen-binding process. Non-cooperative Dioxygen Binding If the oxygen-binding sites M are mutually independent and noninteracting, as in moderately dilute solutions of monomeric molecules, then the concentration of species MO2 as a function of the partial pressure of O2 is generally well fit by a Langmuir isotherm with the fractional saturation of dioxygen binding sites, as Such binding, where the dioxygen sites are independent of each other, is termed non-cooperative. 151 Cooperative Dioxygen Binding Many dioxygen-binding proteins are not independent monomers, with only one dioxygen-binding site, but oligomeric species with the protein comprising two or more similar subunits. The subunits may be held together by van der Waals' forces or by stronger interactions, such as hydrogen bonds or salt bridges, or even by covalent bonds. For example, most mammalian hemoglobins are tetramers where either none, one, two, three, or all four sites may be occupied by dioxygen (Figure 32). Thus, the binding or release of dioxygen at one site may affect the affinity and kinetics of ligand binding and release at a neighboring site. As a result, the saturation curve becomes sigmoidal in shape (Figure 33). When cooperativity is positive, the affinity of a vacant site is increased by occupancy of an adjacent one. 152 Figure 32. Diagram of tetrameric hemoglobin, showing statistical weights of different saturations 153 Figure 33. Binding curves of cooperative and non-cooperative binding of dioxygen. Dioxygen reactions Molecular dioxygen in its ground state is shown below (Figure 34). The bond order for this configuration is 2, containg one and one bond. Hund's rule of multiplicity dictates that the two electrons in the 2p* orbitals occupy different degenerate orbitals. Since they are in different orbitals they will have parallel spins and a net spin angular momentum (that will be in a triplet state). 154 Figure 34. MO energy diagram for a dioxygen molecule. Chemical reactions forming singlet molecules from triplet and singlet reactants are forbidden by Wigner's spin selection rule. Therefore the number of unpaired electrons must remain the same before and after each elementary step of a chemical reaction. For these reasons, is impossible for the following reaction to go in one fast, concerted step: ½ 3O2 + 1X 1XO 155 The arrows represent electron spins: represents a singlet molecule with all electron spins paired; represents a triplet molecule with two unpaired electrons; and represents a doublet molecule, also referred to as a free radical, with one unpaired electron. Majority of the organic compounds are characterized by co-valent bonds where all the electrons are pared (singlet state). Thus, the triplet multiplicity is the actual reason, why most reactions of oxygen with organic substances, although being exergonic, do not proceed at room temperature. It is said that reactions of organic compunds with oxygen are kinetically inhibited. This effect enables our life in an oxygen containig atmosphere. The pathways that do not violate the spin restriction are all costly in energy, resulting in high activation barriers. For example, one can suppose the formation of the excited triplet state of the oxygenated organic product which is followed by a slow spin conversion to a singlet product: ½ 3O2 + 1X 3XO 3 XO 1XO (slowly) Another pathway in which O2 is excited to a singlet state that then reacts with the substrate would be spin-allowed but require a high activation energy: O2 + 22.5 kcal/mol 1O2 3 156 ½ 1O2 + 1X 1XO One pathway for a direct reaction of triplet ground-state dioxygen with a singlet ground-state organic substrate that can occur readily without a catalyst begins with the one-electron oxidation of the substrate by dioxygen. The products of such a reaction would be two doublets, i.e., superoxide and the one electron oxidized substrate, each having one unpaired electron. These free radicals can diffuse apart and then recombine with their spins paired. Such a mechanism has been shown to occur for the reaction of dioxygen with reduced flavins: However, this pathway requires that the substrate be able to reduce dioxygen to superoxide, a reaction that requires an 157 unusually strong reducing agent (such as a reduced flavin), since dioxygen is not a particularly strong one-electron oxidizing agent. The result of these kinetic barriers to dioxygen reactions with most organic molecules is that uncatalyzed reactions of this type are usually quite slow. Most biological conversions involving dioxygen require enzymatic catalysis. The functions of the metalloenzymes for which dioxygen is a substrate are, therefore, to overcome the kinetic barriers imposed by spin restrictions or unfavorable oneelectron reduction pathways, and, for the oxygenase enzymes, to direct the reactions and make them highly specific. Transition metals in the correct oxidation state can react directly with triplet O2 to form dioxygen adducts that can participate in reaction pathways leading to the incorporation of oxygen into organic substrates. Dioxygen, in this case, is not only coordinated, but also activated and made available to the substrate. In binding to metals, O2 effectively functions both as a acid, accepting into its *-orbitals electron density from the filled d-orbitals of the metal, and as a -donor, donating electron density into an empty metal d-orbital (Figure 35). Figure 35. Types of interaction of the electron density of O2 with d-orbitals of iron. 158 The metal d-orbitals, now having partial porphyrin * character, are split, as shown in Figure 36. Figure 36. d-Orbital splitting of Fe3+ ions in metalloporphyrins as a function of number and ligand-field strength. Iron(II) tends to exist in a high-spin configuration where unpaired electrons exist in Eg antibonding orbitals. Iron(III) has an odd number of electrons, and thus must have one or more unpaired electrons, in any energy state. All of these known structures are paramagnetic (have unpaired electrons), not diamagnetic. Thus, a non-intuitive (e.g., a higher-energy for at least one species) distribution of electrons in 159 the combination of iron and oxygen must exist, in order to explain the observed diamagnetism and no unpaired electrons. The three logical possibilities to produce diamagnetic (no net spin) Hb-O2 are: 160 Low-spin Fe2+ binds to singlet oxygen. Both lowspin iron and singlet oxygen are diamagnetic. However, the singlet form of oxygen is the higherenergy form of the molecule. Low-spin Fe3+ binds to O2- (the superoxide ion) and the two unpaired electrons couple antiferromagnetically, giving diamagnetic properties. Low-spin Fe4+ binds to peroxide, O22-. Both are diamagnetic. CHAPTER VI Bioinorganic Chemistry of dinitrogen fixation Nitrogen is a critical limiting element for plant growth and production. It is a major component of chlorophyll, the most important pigment needed for photosynthesis, as well as amino acids, the key building blocks of proteins. It is also found in other important biomolecules, such as ATP and nucleic acids. Even though it is one of the most abundant elements (predominately in the form of nitrogen gas (N2) in the Earth’s atmosphere), plants can only utilize reduced forms of this element. Plants acquire these forms of “combined” nitrogen by: 1) the addition of ammonia and/or nitrate fertilizer (from the Haber-Bosch process) or manure to soil, 2) the release of these compounds during organic matter decomposition, 3) the conversion of atmospheric nitrogen into the compounds by natural processes, such as lightning, and 4) biological nitrogen fixation Nitrogen fixation is the biogenic and non-biogenic transformation of elemental N2 into nitrogen compounds (Figure 37), affording to overcome the bonding energy between the two trebly bonded nitrogen atoms (949 kJ/mol). Nitrogen fixation is a key reaction of the biological nitrogen cycle. The biogenic fixation, carried out by free living nitrogen-fixing bacteria (Azotobacter) and cyanobacteria (“blue-green algae”, Anabaena), some archaea, and by symbiotic bacteria associated with leguminous plants (Rhizobium), leads to ammonium ions. Biogenic fixation accounts for about 60% of the overall nitrogen supply. 161 Figure 37. The nitrogen cycle. The N2 molecule has a triple bond with energy 225 kcal/mole. The challenge to which nitrogenase rises is to break and reduce at a reasonable rate the extremely strong N≡N triple bond. The kinetic inertness of N2 is highlighted by the fact that carrying out reactions "under nitrogen" is considered equivalent to doing the chemistry in an inert atmosphere. Despite this kinetic inertness, thermodynamically the reduction of N2 by H2 is a favorable process, and at pH 7 the reaction 162 has an E0 value that makes it easily accessible to biological reductants such as the low-potential ferredoxins. The thermodynamically favorable reduction of N2 to 2NH3 is a six-electron process. Unless a concerted 6e , 6H + process can be effected, intermediates between N2 and NH3 must be formed. However, all the intermediates on the pathway between N2 and NH3 are higher in energy than either the reactants or the products (Figure 38). Figure 38. Energetics of N2, NH3, and some potential intermediates along the reaction pathway for their interconversion. Several factors may allow this barrier to be overcome. First, the six-electron reduction might be carried out in a 163 concerted or near-concerted manner to avoid the intermediates completely. Alternatively, the intermediates could be complexed at metal centers to stabilize them to a greater extent than either the reactants or products. Finally, the formation reaction for the unfavorable intermedi ate could be coupled with ATP hydrolysis or with the evolution of dihydrogen, each a favorable process, so that the overall process is favorable. The Mo-nitrogenases The action of the Mo-nitrogenase enzyme involves the functioning of two separately isolatable component proteins, as sketched in Figure 39. Figure 39. Organisation of nitrogenase (top), and the structure of the M cluster (bottom). The electrons necessary for the reduction of dinitrogen are delivered by an iron protein containing a cubanelike [4Fe,4S] ferredoxin. Primary e- acceptor is the P cluster of the FeMoco, 164 the ironmolybdenum-cofactor. Two ATP (activated by Mg2+) are hydrolysed per electron transferred. The FeMoco contains two P and two M clusters, arranged in such a way that the complete cofactor attains C2 symmetry. The P cluster is a double cubane containing the Fe8S7 core. The reduction equivalents are then further transported to the M cluster, a Fe7MoS9 core, which is responsible for the final activation and reduction of N2. The cage formed by the metal centres of the M cluster contains electron density which can be interpreted in terms of a nitrogen atom. The M cluster is connected to the protein matrix by just one Cys and a His, the latter coordinated to Mo. The coordination environment of Mo is supplemented by the vicinal hydroxide and carboxylate of homocitrate. In which way activation and reduction of N2 takes place is unknown. The properties of some representative nitrogenases are arranged in the Table 5. Table 5. Properties of some representative nitrogenases. 165 The V-nitrogenases In the case of insufficient molybdenum supply, or at low temperatures, a vanadium-nitrogenase is activated (which is more efficient at lower temperatures than the Mo version). The isolated enzyme was reported to be similar to the Mo enzyme, but had a lower activity and an altered substrate specificity. One of the two components of the V-nitrogenase system is extremely similar to the Fe protein of nitrogenase. Both Fe proteins have an 2 subunit structure, and contain a single Fe4S4 cluster. A major difference between the V and Mo enzymes lies in substrate specificity and product formation. the FeV nitrogenase has a much lower reactivity toward acetylene than does the Mo system. Furthermore, whereas the FeMo system exclusively produces ethylene from acetylene, the FeV system yields significant amounts of the four-electron reduction product, ethane. The all-iron nitrogenase The first sign that there is yet another alternative nitrogenase again came from genetic studies. The finding of the all-iron nitrogenase, if fully confirmed, will add significantly to the comparative biochemistry of nitrogen fixation. Speculatively, one might suggest that the concomitant absence of V and Mo suggests that nitrogen fixation need not directly involve the noniron heterometal in the cofactor cluster. 166 Model systems Three types of model systems for nitrogenase may be considered. First, there are transition-metal sulfide clusters that resemble the FeMoco or FeVco centers of the active proteins. Both X-Ray structural analysis on single crystals, and quantumchemical modeling of the enzyme active sites are used in order to predict the possible structures of intermediate complexes (Figure 40). Figure 40. Dinitrogen bound intermediate of the active site of nitrogenase from DFT calculations. A second approach uses the reactions of N2 and related substrates or intermediates with metal centers in order to gain insights into the way in which transition-metal systems bind N2 and activate it toward reduction. As an example, in 2011 Arashiba et al. reported yet another system with a catalyst again based on molybdenum but with a diphosphorus pincer ligand: 167 And finally, there are other inorganic systems that display some of the structural and possibly some of the reactivity characteristics of the nitrogenase active sites without binding or reducing N2 or precisely mimicking the active center. 168 EXPERIMENTS In Bioinorganic Chemistry 169 170 EXPERIMENT 1 Electronic structures of atoms and ions. Molecular diagrams of molecules and molecular ions. Bond orders and multiplicities of electronic states Goals. To understand how electronic configurations of atoms and molecules affects their possible valence states and configurations of reactional centers of biomolecular complexes. Theoretical part. In atomic physics and quantum chemistry, the electronic configuration is the distribution of electrons of an atom or molecule (or other physical structure) in atomic or molecular orbitals. According to the laws of quantum mechanics, an energy is associated with each electron configuration and, upon certain conditions, electrons are able to move from one orbital to another. Knowledge of the electronic configuration of different atoms and molecules is useful for describing the chemical bonds of different classes of chemical compounds. Experimental part. 1. Draw the electronic configurations of the following atoms and ions: Zn and Zn2+; Cu, Cu+ and Cu2+; Co, Co2+ and Co3+; Fe, Fe2+, Fe3+ and Fe4+; Mo, Mo2+ and Mo6+; V, V2+, V3+ and V5+. For each group compare the relative stabilities of species. Indicate the number of unpaired electrons. 2. Draw the electronic configurations of phosphorus, sulfur and oxygen in the ground and possible excited states. Predict 171 possible valence states of the above elements and present the formulae of the corresponding compounds. 3. Using the VB method, describe the structures of the following molecules and ions: H2O, H3O+, NH3, NH4+. Indicate the type of hybridization of atoms and their spatial disposition. 4. Explain in terms of the VB theory the following observations: (a) CCl4 and SiCl4 are both tetrahedral; (b) CO2 is linear but SO2 is angular; (c) HSH angle in H2S is closer to 90 than the HOH angle in H2O; (d) PCl5 exists but not NCl5; (e) BF3 is planar but NH3 is not. 5. The valence electron configuration of Hg is 6s2. What kind of hybrid orbitals can be used to account for the linear geometry of HgCl2? 6. Use the MO energy level diagrams to determine bond orders and multiplicities of the ground states of the following molecules and ions: O2, O2-, O22-, N2, N22-. 7. Which of the following pairs of molecules would you expect to have the higher bond energy: (a) F2 and F2+; (b) NO and NO-; (c) BN and BO; (d) NF and NO? 8. Explain in terms of the MO theory the following observations: (a) the first ionization energy of NO is less than that of CO; (b) the first ionization energy of O2 is less than that of the oxygen atom. 172 EXPERIMENT 2 Electronic and spatial structures and chemical properties of coordinate compounds Goals. To study the reactions of synthesis, decomposition, and exchange in the inner spheres of coordinate compounds. To correlate colors of the coordinate compounds of the transitional elements with the strengths of the ligand field. Theoretical part. A coordination complex or metal complex, consists of an atom or ion (usually metallic), and a surrounding array of bound molecules or anions, that are in turn known as ligands or complexing agents. Many of the properties of metal complexes are dictated by their electronic structures. The electronic structure can be described by a relatively ionic model that ascribes formal charges to the metals and ligands. This approach is the essence of crystal field theory (CFT). Crystal field theory, introduced by Hans Bethe in 1929, gives a quantum mechanically based attempt at understanding complexes. More sophisticated models embrace covalency, and this approach is described by ligand field theory (LFT) and Molecular orbital theory (MO). They can handle a broader range of complexes and can explain complexes in which the interactions are covalent. 173 Experimental part. I. Formation of complex compounds. 1. Put 4-5 drops of the aqueous solution of copper chloride into a test tube. Add ammonium hydroxide until the precipitate is formed. Add excess of NH4OH and observe the formation of a complex compound. Indicate the change in color while transformation of an aqua-complex of copper(II) into the ammine complex. Draw the electronic formula of the Cu2+ cation. Using the VB method, determine the type of hybridization of the central atom in the ammine complex. Draw the MO energy level diagrams for the squaric Cu(II) complexes. Basing on the colors of the complexes, compare the ligand field strengths of the water and ammonia ligands. 2. Put 4-5 drops of the aqueous solution of nickel chloride into a test tube. Add Add 1 ml of the dimethylglyoxime solution and observe the formation of a complex compound. Indicate the change in color while transformation of an aqua-complex of nickel(II) into the dimethylglyoximato-complex. Draw the electronic formula of the Ni2+ cation. Draw the MO energy level diagrams for the octahedric Ni(II) complexes. Basing on the colors of the complexes, compare the ligand field strengths of the water and dimethylglyoxime ligands. II. Destruction of complex compounds. 1.Take the solution of chloride tetraammine copper(II), obtained from the previous experiment, add dropwise diluted (1:1) nitric acid until the complex compound is destroyed. Indicate the change in color. Write the reaction in a net ionic form. 174 2.Take from 5 to 6 drops of CdCl2 and HgSO4 into sepaate test tubes. Add dropwise a saturated solution of Na2SO3. Observe the formation of precipitates of the corresponding sulfites and their further transformation into complex compounds of the composition of Na2[M(SO3)2], M= Cd2+ or Hg2+. Add the solution of NaOH to both the solutions of complex compounds and notice your observatins. Compare the instability constants of Na2[Cd(SO3)2] and Na2[Hg(SO3)2] with the solubility product constants of the corresponding metal hydroxides and conclude about the possibility of the metal complexes to be destroyed by the solution of NaOH. III. Exchange of ligands in complex compounds. 1.Take 5 to 6 drops of the solution of FeCl3 in a test tube and add 2 to 3 drops of the solution of NH4SCN. Observe the change in color while complex formation. Then add some crystals of NH4F and shake well the test tube. Write down your observations. Compare the instability constants of the complex ions and conclude about the possibility of the reaction of ligand exchange. Draw the electronic structures and determine the types of hybridization if a ferric cation in the complexes of the ligands of a weak and a strong field. 2.Perform the corresponding calculations and prove experimentally that SCN- ligands can not replace fluoride anions from the inner sphere of the ferric complex. 175 EXPERIMENT 3 Redox-properties of complexes of transition metals Goals. To study the redox-activities of central ions in coordinate compounds. Theoretical part. Complex compounds may possess redox-properties and be involved in the reactions where the oxidation state of the central ion changes. Two types of redox reactions of complex compounds are possible. i) The oxidation state changes without decomposition of the inner sphere: 2K3[Fe3+(CN)6] + 2KI = 2K4[Fe2+CN)6] + I2 ii) The change in the oxidation state of the central atom provokes the destruction of a complex compound: Zn + 2K[Au+(CN)2] = 2Auo + K2[Zn(CN)4] Experimental part. I. Oxidation of the central ion. 1. Take 1 ml of the solution of KMnO4 in a test tube, add an equal volume of a diluted solution of H2SO4 and then drpowise K4[Fe(CN)6]. Observe the change in color of the solution. Prove the formation of K3[Fe(CN)6] (probe with FeSO4 solution). If necessary, peform test reactions of K4[Fe(CN)6] and K3[Fe(CN)6] with the solutions of Fe(II) and 176 Fe(III) salts. Write and balance the reaction of oxidation of K4[Fe(CN)6] into K3[Fe(CN)6] by KMnO4 in the acidic medium. 2. Take 1 ml of the solution of CoCl2 and 2 ml of ammonia solution in one test tube. Indicate the color of the ammine complex of Co(II). Add 1 ml of a NH4Cl solution and 2 ml of H2O2. Heat the test tube and indicate the change in color because of oxidation of Co(II) into Co(III). Add H2O2 to the neutral solution of CoCl2 in another test tube (do not add NH3 solution). Heat the solution and compare oxidating abilities of Co2+ cations in [Co(H2O)6]2+ and [Co(NH3)6]2+ complexes. Write and balance the redox-reaction. II. Reduction of the central ion. 1. Take 1 ml of the solution of CuSO4 in a test tube and add dropwise 25% solution of NH3 until the formation of a darkblue solution of [Cu(NH3)4]SO4. Put a zinc granule into the solution and observe the changes on the surface of the granule. Write and balance all the the reactions. 2. Mix equal volumes of AgNO3 and NaCl solutions (10 to 12 drops each) in a test tube. Dissolve the formed precipitate in the concentrated ammonia solution. Add the Zn granule to the solution. Write down and explain your observations. Write and balance all the the reactions. III. Redox reactions while complex formation. Take 5 to 6 drops of any Co(II) salt solution into a test tube and add 5 drops of CH3COOH and the same amount of KNO2. Observe the formation of a yellow precipitate of K3[Co(NO2)6]. Is the studied reaction a redox one? Write and balance it. 177 EXPERIMENT 4 Quantum-chemical modeling of tautomeric forms of nucleic acids and their complexes with metals Goals. Application of methods of quantum chemical modeling for prediction of tautomeric isomerism of organic species and their complexes with metals. Theoretical part. Computational Methods for Bioinorganic Systems are widely used. Proteins contain many atoms, too many to all be treated using accurate electronic structure methods based on quantum mechanics (QM) using current methods and computers. As a result, people have developed hybrid "QM/MM" methods in which the system is partitioned into a reactive "core" which is treated using QM, and a remaining "environment" which is treated using the much less expensive molecular mechanics (MM). A variety of such QM/MM methods, depending on the type of QM and MM used, and the way in which the interaction of the QM and MM regions is treated. Potential energy surfaces for proteins are also extremely complicated, with many similar minima differing in the orientation of amino acid sidechains, water molecules, etc. For the nowdays, a large variety of computer packages of programs for quantum chemical modeling purposes exist. The mostly used are MOPAC, ChemOffice, HyperChem, Gamess and Gaussian. A semiempirical PM3 method is proposed for calculation of relative stabilities of tautomers and conformers of organic 178 molecules and their complexes with metals. All the calculations should be performed with the Gamess complex. The ChemOffice package is to be used for construction and redaction of structures, and for the visualization of the results the ChemCraft program is proposed. Experimental part. 1.Chose the object of investigation out of the proposed structures (nucleic bases or amino acids). 2.Using the ChemOffice package, draw all possible tautomeric forms of the molecule to be studied. 3.Optimize geometry using molecular mechanics approach and then perform quantum chemical geometry optimization. 4.Save the coordinates of optimized structure into a file; show the optimized bondlengths and valence angles using preferred visualisation software (e.g. ChemCraft). Write down the heat of formation and the total energy of the obtained conformer. 5.Repeat the procedure presented in points 3 and 4 for the other possible tautomeric forms of the compound. 6.Basing on the calculated H or Etotal values of the tautomers, conclude on their relative stabilities. Compare the calculated results with the literary data on the form of existence of the studied substance in crystalline form and in solutions. 7.Construct all the possible structures of the complexes of the studied molecule with one of the metals (consult your instructor). Pay attention to monodental or chelating coordination and all possible active sites of the organic species. 8.Repeat the procedure presented in points 3 – 6. Make your conclusion on the most probable structure of the complex compound. 179 EXPERIMENT 5 Determination of ionization constants of amino acids by potentiometry Goals. Application of physico chemical methods (potentiometry) for determination of ionization constants of organic species. Theoretical part. Potentiometry involves measuring an electrical potential that is related to a component in which someone is interested. For example, many communities fluoridate water supplies to enhance the durability of tooth enamel. One convenient way to measure fluoride concentration is to use an electrode where the potential depends upon the concentration of fluoride. Potentiometric electrodes are usually fast, portable, and do not require extensive training to operate. The most common electrode in use is the glass membrane pH electrode (see figure). The difference in electrical charge across the glass membrane is the membrane potential that depends only on the concentration of H3O+ in the outer solution. Organic acids usually possess weak properties, i.a. their dissociation is not full and reversible; a dynamic equilibrium exists, which is represented by the following equation: HA + H2O H3O+ + A– The equilibrium expression for this reaction is: 180 Ka [ HA] [ H 3O ][ A ] or [ H 3O ] K a [ HA] [A ] (1), where Ka is the acid-ionization constant for the weak acid. Let us assume that the initial dissociation of the weak acid is negligible. The progressive addition of NaOH during the titration decreases the concentration of HA and increases the concentration of its salt, NaA: HA (aq) + NaOH (aq) H2O(l) + NaA (aq) The presence of both HA and its salt, NaA, creates a buffer system, which resists a large change in pH. The ratio of [HA]/[A–] changes only slightly; therefore, according to (1), the change in [H3O+] (or pH) must also be small. The pH increases slowly until the equivalence point is approached: At the halfway point in the titration, exactly half of the HA originally present will have been neutralized, and therefore the concentrations of HA and A– will be equal. [ H 3O ]1 / 2 [ A ]1 / 2 ; Ka [ HA]1 / 2 K a [ H 3O ]1 / 2 (2) 181 Thus, the ionization constant of a weak acid is equal to the hydronium ion concentration at the halfway point in the titration; pKa = pH1/2. This relationship is valid only if the initial dissociation of the acid is negligible. When the degree of dissociation is appreciable, as in the case of a very dilute solution, the pH at the midpoint of the titration bears no relation to the value of Ka. Amino acids are a little different from the other weak acids. When the amino acids are placed in a strong acid like HCl, as will be the case for this experiment, the amino acid will be completely protonated and have a net ionic charge of +1. As the base is added in the titration, the proton on the carboxylic acid will be removed first, so that around a pH of 7, the zwitterionic form will exist. At the end of the titration, the amino acid will be completely deprotonated.and have a charge of -1: pH 1 Net charge +1 pH 7 Net charge 0 O R pH 13 Net charge -1 O O OH NH3+ Cationic form R O- NH3+ Zwitterionic form R OH NH2 Anionic form A titration curve of aminoacids starting from strongly acidic solutions therefore looks like: 182 Experimental part. 1. Prepare the necessary reagents and equipment: Chemicals Equipment 1 M HCl Weighing boats 0.1 N NaOH Spatula Amino acid (assigned to each student) pH 4 and 7 buffers Electronic pan balance 1-calibrated pH Checker Ring stand Buret and clamp 125 ml Erlenmeyer flask 2. Mass out a sample of your amino acid (not more than 0.1000 g) and record the name of the amino acid and its mass. 3. Pour the sample into the pre-labeled Erlenmeyer flask and dissolve the amino acid in 25 ml of water. 4. Obtain your pH checker and turn it on. Insert it into the amino acid solution and allow it to stabilize. Once it has stabilized, add enough 1 M HCl to it to adjust the pH of the solution to a pH of about 1 to 1.5. Record that pH and the volume of HCl added. 5. Obtain and clean a 50 ml buret. Condition the buret with a standardized sodium hydroxide solution of concentration approximately 0.1 M. Place the buret in a buret holder on a ring stand and fill with the sodium hydroxide solution. Make sure that there are no bubbles neither in the buret nor in its tip. Record the initial volume reading in your notebook showing two decimal places. 6. Add stepwise 0.5 ml of the NaOH solution to the studied solution while stirring, and record pH after each addition (in total 50 ml of NaOH should be added). Arrange your results as a table: V(NaO 0 0. 1. 1. 2. … … … … … … … H), ml 5 0 5 0 pH 183 7. Using the results of the titration, set up the titration curve (pH vs. V(NaOH)) and first-derivative curve (pH/V vs. V(NaOH)) (see the figure above). 8. For pKCOOH determination, find the first endpoint volume (from your first derivative curve) and divide that in half. Record the experimental pKCOOH value. 9. For pKNH2 determination, find the first and second endpoint volumes from your first derivative curve. Find the halfway volume between the two. Back determine the pKNH2 at that volume from your titration curve. Record the experimental pKNH2 value. 10. Insdead of points 7 – 9, you can also use a Hyperquad computer program for calculations. 11. Search the pKCOOH and pKNH2 values in the internet databases and calculate the error of your experiment. 184 EXPERIMENT 6 Determination of stability constants of complexes of transitional metals by potentiometry Goals. Application of physico chemical methods (potentiometry) for determination of stability constants of complexes of metals with organic species. Theoretical part. General approach. Metal ions in solutions most often exist as solvates (hydrates in aqueous solutions). The reaction of complex formation may be considered as the reaction of ligand exchange: М(Н2O)n + L → M(H2O)n-1,L + Н2O where М is a metal ion, and L is a ligand. During the complex formation, the coordinated (hydrated) water molecules are consistently replaced by ions or molecules of ligands to form MLn complex (n here indicates number of ligand molecules). For easier presentation, the solvated metallic ion can be presented in a “free” form (Мe+x). The reaction of complex formation can be presented as: mM+x + nL→MmLn The thermodynamic constant of this reaction can be expressed as following: where f are activity coefficients. 185 In the case when the ionic strength I lies in the interval 0.1 – 0.5, the concentrational overall stability constant may be used: The complex formation usually occurs stepwise: M + L↔ML1 ML1 + L↔ML2 …………………….. ……………………... MLn-1 + L ↔ MLn Stepwise stability constants are determing as following equations: The product of stepwise stability constant is known as an overall formation constant: β = К1·К2·...Кn Potentiometric determinations. A lot of methods of determination of stability constants of complex compounds exist, and the method of potentiometric titration is one of them. In this method, the ligand possessing acidic properties is titrated by an alkali solution individually and then in its mixture with a metal salt. As hydrogen ions of the ligand (weak acid) are replaced by a metal according to the equation: M + xHL↔MLx + xH+, the titration curve of the metal-to-ligand mixture will lie in a lower pH range than the one of the individual ligand: 186 This difference in titration curves gives the possibility to calculate the formation constant of a comples using potentiometric titration and a special characteristic of the equilibrium process of complex formation which is called _ formation function, n : C ( HL ) [ L ] n , C (M ) _ where C(HL) an C(M) are total ligand and metal concentrations, and [L-] is the equilibrium concentration of the non-complexed ligand. The formation function indicates the mean ligand molecules bound to one metal ion. For a chelating ligand (i.e. aminoacid): log([L-]) = log(C(HL) – C(NaOH) – [H+] + [OH-]) – logP [ H ] 2[ H ] 2 P where K2 > K1. K2 K1 K 2 187 In the case of formation of a 1 : 2 complexes of metals with amino acids _ n 2C ( NaOH ) C ( HL ) The total concentration of the amino acid includes deprotonated, neutral, protonated, and coordinated species: C(HL) = [L-] + [HL] + [H2L+] + [ML] + 2[ML2]; _ [ ML ] 2[ ML2 ] n ; C (M ) _ C(HL) = [L-] + [HL] + [H2L+] + n C(M); and finaly, _ _ K1 n _ ; (1 n) [ L ] K2 n 1 _ ; = K1K2. ( 2 n) [ L ] Experimental part. 1. Prepare the necessary reagents and equipment: Chemicals Equipment 1 M HCl Weighing boats 0.1 N NaOH Spatula Amino acid (assigned to each Electronic pan balance student) Metal salt (assigned to each Buret and clamp student) pH 4 and 7 buffers Ring stand 1-calibrated pH Checker 125 ml Erlenmeyer flask 188 2. Mass out a sample of your amino acid (the same name and the same mass as in the previous experiment) and record the name of the amino acid and its mass. Calculate the number of moles of the sample. 3. Considering that a ML2 complex is formed, calculate the necessary amount and mass out a sample of the metal salt. 4. Pour the samples of both metal salt and amino acid into the pre-labeled Erlenmeyer flask and dissolve the mixture in 25 ml of water. 5. Obtain your pH checker and turn it on. Insert it into the amino acid solution and allow it to stabilize. Once it has stabilized, add enough 1 M HCl to it to adjust the pH of the solution to a pH of about 1 to 1.5 (same value as in the previous experiment). Record that pH and the volume of HCl added. 6. Obtain and clean a 50 ml buret. Condition the buret with a standardized sodium hydroxide solution of concentration approximately 0.1 M. Place the buret in a buret holder on a ring stand and fill with the sodium hydroxide solution. Make sure that there are no bubbles neither in the buret nor in its tip. Record the initial volume reading in your notebook showing two decimal places. 7. Add stepwise 0.5 ml of the NaOH solution to the studied solution while stirring, and record pH after each addition (in total 50 ml of NaOH should be added). Arrange your results as a table: V(NaOH), 0 0.5 1.0 1.5 2.0 … … … … … ml pH 8. Using the results of the titration, set up the titration curve (pH vs. V(NaOH)) and add the titration curve obtained from the previous experiment (see the figure above). 189 log*** logK2*** _ n *** logK1*** log[L-]** C(NaOH), mol/l* C(M), mol/l* C(HL), mol/l* pH V(NaOH), ml No of the experiment 9. Basing on the mutual disposition of the two curves, conclude on the ionic form of the ligand in the complex (neutral or deprotonated). 10. Use the Hyperquad computer program to calculate stepwise stability constants K1 and K2, and the overall formation constant . You may also use the algorithm presented in the table below: 1 2 3 4 5 6 7 8 9 10 1 2 ... ... * the dilution should be taken into consideration: V (taken) ; С С (initial ) V (total) ** log[L-] = (pH – pK(COOH) + log(C(HL) – C(NaOH)); for pK(COOH) see the previous experiment; *** see the theoretical part. 11. Calculate the mean K1, K2 and values and mean deviations using mathematical statistics methods. 190 EXPERIMENT 7 Determination of protonation constants of nucleic bases by UV-spectrophotometry Goals. Application of physico chemical methods (UVspectrophotometry) for determination of protonation constants of organic species. Theoretical part. General approach. Spectrophotometry is one of the most important practices carried out in laboratories. Spectrophotometry is the study of the reflection or transmission properties of a substance as a function of wavelength. It consists of the qualitative and quantitative studies. The energy in the UV and visible regions ( 200 – 700 nm) irradiated on the molecules can result in changes in the electronic nature of the molecule i.e. changes between ground state and excited states of electrons within the system. As a result, UV-visible spectroscopy is also known as electronic spectroscopy. As it was shown previously (see Lecture 1), the electrons in the molecules occupy so called molecular orbitals (MO). The electron transitions occur between occupied and unoccupied MOs. The energy of the electron transition (i.e. E between the occupied and unoccupied orbitals) is in accordance to the absorbed wavelength: 191 R here is a Rydberg Constant (1.0974x107 m-1); is the wavelength; n is equal to the energy level (initial and final). The electronic transitions in organic compounds can be determined by ultraviolet-visible spectroscopy, provided that transitions in the ultraviolet (UV) or visible range of the electromagnetic spectrum exist for this compounds. The longwawe absorption refers to the electron transition from the higest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). The change of the electronic structures of the molecules because their tautomeric transformations, ionization (protonation or deprotonation), complexation, ets. leads to the changes in energies of HOMO and LUMO and, as a result, shifting of the transition bands in the spectra. Quantatively, the absorbance of light by a substance is described by the Beer–Lambert–Bouguer law. It states that there is a logarithmic dependence between the transmission (or transmissivity), T, of light through a substance and the product of theabsorption coefficient of the substance, α, and the distance the light travels through the material (i.e., the path length), ℓ. The absorption coefficient can, in turn, be written as a product of either a molar absorptivity (extinction coefficient) of the absorber, ε, and the molar concentration С of absorbing species in the material: , where 0 and are the intensity (or power) of the incident light and the transmitted light. The transmission (or transmissivity) is expressed in terms of an absorbance which, for liquids, is defined as 192 or A Cl . Spectrophotometric titration. Spectrophotometric titration is an analytical method in which the radiant-energy absorption of a solution is measured spectrophotometrically after each increment of titrant is added. The studies of equilibrium processes are based on the difference in absorption spectra of two forms of a substance (for example, its neutral and ionized form). The existence of the equilibrium is proved by the presence of one or several isobestic points: Isosbestic point is a specific wavelength at which two chemical species have the same molar absorptivity (ε) or, more generally, are linearly related. The requirement for an isosbestic 193 point to occur is that the two species involved are related linearly by stoichiometry, such that the absorbance is invariant for one particular wavelength. The presence of an isosbestic point typically does indicate that only two species that vary in concentration contribute to the absorption around the isosbestic point. In case if an acid-base equilibrium of a weak acid or a weak base is studied, it is evident that in acidic media a protonated base or a neutral form of an acid are present in the solution. The alkaline media are related to the neutral form of a base and deprotonated form of an acid. Thus, while titration of acidified solutions of a weak base or a weak acid, the following processes takes place: HA H+ + AHB+ H+ + B The equilibria constants of the processes can be expressed as Ka [ H ][ A ] ; [ HA] Kb [ H ][ B] ; [ HB ] In general, if is considered as the degree of dissociation, K [H ] . 1 The value at the each titration moment can be calculated from the absorbance at a given pH. If the absorbance in the acidified solution relates to the non-dissociated particles (=0), and the absorbance in the alkaline solutions relates to their full dissociation (=1), so A Aacidic A Aacidic ; ; K [H ] Abasic Aacidic Abasic A 194 pK pH log Abasic A A Aacidic Experimental part. 1. Prepare the necessary reagents and equipment: Chemicals Equipment 1 M HCl Weighing boats 0.1 N NaOH Spatula Ethanol Electronic pan balance Nucleic base (assigned to each Macro (25 ml) and student) micropipets pH 4 and 7 buffers 50 ml beaker 1-calibrated pH Checker 50 ml measuring flask Magnetic rotator UV-VIS spectrophotometer 2. Mass out a sample of your nucleic base and prepare 50 ml of a 10-3 M solution in ethanol. 3. Record the UV-Vis spectrum of the sample according to the common procedure (see the Instructions for the available spectrophotometer). Dilute the solution if nessary to obtain a good spectrum. Write down the necessary concentration. 4. Take 25 ml of the prepared solution in a beaker and measure its pH. Add HCl dropwise until pH 1 is adjusted. 5. Adjust your spectrophotometer program for spectrophotometric titration (see the Instructions). 6. Record the spectrum of the acidified solution. (Return the solution back to the beaker after each spectrum recordance). 195 7. Add 1 drop of the NaOH solution, stir it carefully with the magnetic rotator, and measure pH (all measured pHs should be added to the table (see below)). Record the spectrum. 8. Repeat the procedure 7 until pH 12 (or until there are no more changes in the spectrum). 9. Save and print the results of the spectrophotometric titration. 10. For calculations, select one or several analytical wavelengths (they require to the maximal changes in absorption within titration). As an example, see the Figure above. 11. Measure absorbances at each titration point and arrange the following table: No of pH A(1) pK A(2) pK … experiment 1 2 … 12. Calculate pK value for all the intermediate points (consider Ainitial as Aacidic and Afinal as Abasic. 13. Calculate the mean K1, K2 and values and mean deviations using mathematical statistics methods. 14. Search the corresponding pK value in the internet databases and calculate the error of your experiment. 196 EXPERIMENT 8 Determination of stability constants of complexes of transitional metals with the nuclear bases by UV-spectrophotometry Goals. Application of physico chemical methods (UVspectrophotometry) for determination of stability constants of complexes of metals with nucleic bases. Theoretical part. In the spectrophotometric method, the molar absorptivity of the complex is an additional variable to be determined. As well, it is necessary to determine the stoichiometry of the complex before calculations can be performed. In the mole-ratio method a series of solutions is prepared in which the concentration of one reactant (usually the ligand) is held constant while the other reactant (usually the metal ion) is varied. The titration of the ligand solution by the solution of a metal salt is performed, and the UV-Vis spectra are recorded after each addition. The absorbance at the analytical wavelength is then plotted versus the concentration of the metal in the solution: 197 If only one complex of high stability is formed, the graph consists of two linear intersecting parts. The ratio of the concentration of ligand at the intersection point to the (fixed) concentration of metal ion gives the stoichiometry of the complex. If the stability constant of the complex is high (curve A), there will be no appreciable dissociation of the complex at or near the stoichiometric point. If the complex is moderately stable (curve B), the plot will consist of two straight-line portions with a central curved portion. Extrapolation of the two straight-line portions yields the intersection point. If a complex of low stability is formed, a large excess of ligand will have to be used to drive the reaction to completion, and there will be no detectable break in the curve from which to obtain the stoichiometry (curves C and D). 198 Once the stoichiometry of the complex has been established, the formation constant can be calculated provided the data yields a curve showing some dissociation in the neighborhood of the stoichiometric point (curve B). Briefly, for any data point in the region of curvature, complex formation did not proceed to completion, as evidenced from the difference between the measured curve (B) and the “theoretical” one (A). Here there is obviously an equilibrium between metal ion, ligand and complex: nMx+ + mL MnLm. The equilibrium constant of this process represents the formation constant : K M n Lm ] . [ M x ] n [ L] n The equilibria concentrations of non-complexed particles [Mx+] = CM – n[MnLm]; [L] = CL – m[MnLm] (CM and CL are total concentrations of the metal ion and the ligand in the equivalent point with respect to the mutual dilution of solutions while mixing). Considering a full transformation of the reactants (“theoretical” curve A), A(complex) = (complex) ℓ [MnLm]0 where [MnLm]0 = 1/n CM. (“theoretical” concentration) So, the value of (complex) is calculated from the curve A. Under the real conditions (experimental curve B), the concentration of the complex [MnLm] < [MnLm]0 . From the experimental curve B which describes the equilibrium process of complex formation, the real concentration of the complex [MnLm] in the solution may be calculated: 199 A(equilibrium) = (complex) ℓ [MnLm] (the (complex) value is already determined from the “theoretical” curve A). Now equilibria concentrations of the metal ion and the ligand can be found as: [Mx+] = CM – n[MnLm]; [L] = CL – m[MnLm]. And finally, knowing the composition of the complex and equilibria concentrations of all the particles in the solution, the formation constant of the complex can be easily calculated: M n Lm ] . [ M x ]n [ L ]n Experimental part. 1. Prepare the necessary reagents and equipment: Chemicals Equipment Ethanol Weighing boats Nucleic base (assigned to Spatula each student) Metal salt (assigned to each Electronic pan balance student) Macro (25 ml) and micropipets 50 ml beaker Two 50 ml measuring flasks Magnetic rotator UV-Vis spectrophotometer 2. Mass out a sample of your nucleic base and prepare 50 ml of a solution of a nesessary concentration (see the previous experiment). 200 3. Mass out a sample of the metal salt and prepare 50 ml of a solution which is 100 times more concentrated than the solution of a nucleic base. 4. Take 25 ml of the prepared solution of the nicleic base in a beaker and record the UV-Vis spectrum according to the common procedure (see the Instructions for the available spectrophotometer). 5. Add stepwise 0.05 ml of the solution of a metal salt and record the spectra after each addition (in total, you should add 1 ml of the solution of a metal salt to 25 ml of the ligand solution). 6. Select one or several analytical wavelengths (see the previous experiment) and measure absorbance of each curve in the titration. 7. Calculate the concentration of a metal salt at each point (consider the dilution of the solution of the metal salt after addition to the ligand). 8. Plot the absorbance at the analytical wavelength versus the concentration of the metal in the solution, extrapolate the two straight-line portions and determine the intersection point. 9. Knowing concentrations of metal and ligand, find the composition of the complex in the solution. 10. Follow the procedure presented in the theoretical part and calculate the stability constant of the complex compound. 201 EXPERIMENT 9 Determination of coordination sites of O- and N-donating ligands by IR-spectroscopy Goals. Application of physico chemical methods (IRspectroskopy) for determination of active sites of organic compounds in the process of complex formation. Theoretical part. Infrared radiation is that part of the electromagnetic spectrum between the visible and microwave regions. Infrared radiation is absorbed by organic molecules and converted into energy of molecular vibration, either stretching or bending. Different types of bonds, and thus different functional groups, absorb infrared radiation of different wavelengths. A IR spectrum is a plot of wavenumber (X-axis) vs percent transmittance (Yaxis). (Note: wavelength can be used instead of wavenumber and absorbance instead of percent transmittance). A molecule or a functional group can vibrate in many ways, and each way is called a vibrational mode. For molecules with N atoms in them, linear molecules have 3N – 5 degrees of vibrational modes, whereas nonlinear molecules have 3N – 6 degrees of vibrational modes. These are symmetric (a) and antisymmetric stretching (b) or bending (c): 202 Complex IR spectra are interpreted by extrapolating from such studies of simple molecules, since most functional groups give rise to bands in particular regions of the spectrum. The region from 4000-1300 cm-1 is particularly useful for identifying the presence of specific functional groups: 3600 –2700 cm-1 for X-H stretchings (C-H; O-H; N-H); 2700 – 1900 cm-1 for X≡Y stretchings (C≡N; C≡C); 1900 – 1500 cm-1 for X=Y stretchings (C=O; C=N; N=N; C=C); and 1500 – 500 cm-1 for X-Y stretchings and X-H bendings. Characteristic IR Absorption Frequencies of Organic Functional Groups Characteristic Functional Type of Absorptions Intensity Group Vibration (cm-1) Hydroxyl O-H (stretch, H-bonded) 3200-3600 strong, broad 203 O-H (stretch, free) 3500-3700 strong, sharp C-O (stretch) 1050-1150 strong Amine N-H stretch 3300-3500 medium (primary amines have two bands; secondary have one band, often very weak) C-N stretch 1080-1360 medium-weak N-H bending 1600 medium C-H stretch 3000-3100 medium C=C stretch 1400-1600 medium-weak, multiple bands Aromatic IR Absorption Frequencies of Functional Groups Containing a Carbonyl (C=O) Functional Type of Characteristic Intensity Group Vibration Absorptions (cm-1) Carbonyl C=O stretch 1670-1820 strong (conjugation and coordination moves absorptions to lower wave numbers) 204 Acid C=O stretch 1700-1725 strong O-H stretch 2500-3300 strong, very broad C-O stretch 1210-1320 strong C=O stretch 1640-1690 strong N-H stretch 3100-3500 unsubstituted have two bands N-H bending 1550-1640 C=O stretch 1735-1750 strong C-O stretch 1000-1300 two bands or more Ketone acyclic stretch 1705-1725 strong cyclic stretch 3-membered – 1850 4-membered – 1780 5-membered – 1745 6-membered – 1715 7-membered – 1705 strong ,unsaturated stretch 1665-1685 strong aryl ketone stretch 1680-1700 strong Amide Ester 205 Experimental part. 1. Prepare the necessary reagents and equipment: Chemicals Equipment Ethanol Weighing boats Amino acid (assigned to each Spatula student) Metal acetate (assigned to each Electronic pan balance student) NaOH (diluted) 100 ml beaker Nuyol Heater KBr or NaCl disks Magnetic rotator IR spectrophotometer 2. Peform the synthesis of the metal complex of one of the amino acids according to the following procedure. Metal(II) acetate (0.01 mole) is dissolved in 25 cm3 of hot water. 25 cm3 of hot ethanol is added and the solution kept hot. Amino acid (0.02 moles) is dissolved in 25 cm3 of hot water. The solutions are mixed while hot (~70C) and the solution then cooled on ice. In the case if no precipitation is observed, the pH of the mixture is adjusted to 7 – 8 with the help of the diluted NaOH solution. After 2 hours, the formed precipitate if filtered of (or centrifugated), washed with a small amount of ethanol and dried in a dessicator over P2O5. 3. Prepare the dried sample of your complex as a suspention in Nuyol between two KBr or NaCl disks and record an IR spectrum according to a general procedure (consult the Instructions for the IR spectrophotometer). 4. Using the procedure 3, record the IR spectrum of the noncoordinated amino acid. 206 5. Examine the IR spectrum of the amino acid. Using the tables presented above, find the absorption bands which relate to vibration modes of the amino- and carboxylic groups (how many bands do you expect to describe and which vibration modes they represent?). 6. Compare the spectra of the complex and the non-coordinated amino acid and make your conclusions about the shifting of the absorbtion bands while coordination. 207 208 Instructions for Research topic: Presentation, Questions and Term Paper During the first week of class, each student enrolled in the course will choose a research topic out of the list included above or propose his/her own topic related to the bioinorganic chemistry for a class handout and presentation, as well as for a term paper. (Note! Your research topic should be approved by the instructor). Because the purpose is to have you learn something new, please choose a topic you have not studied in a previous course. The results of your researches should be arranged as a Term Paper and Presentation. The volume of the Term Paper should not exceed 10 – 15 pages. It should consist of the background information, research problems, discussion, and conclusions. Include at least one figure or table containing experimental data from your research topic, and explain the interpretation of the data in the figure or table. The list of citations should contain not less than 5 references: books or journal papers. avoid citations to web sites unless you cannot find the information in a refereed journal article (and in such a case make sure it is a trusted web site such as one of those suggested by TEXT). The class presentation should be a 10-minute “chalk talk” accompanied by slides or a poster.The presentation and paper may both start from the same outline, but because of the brief time allocated to your presentation, you will need to condense the background information and omit other recent results in your presentation: a) What is the biological function and/or medical relevance of the system? In what species is this system found, and 209 from which species is the system best characterized? If this a protein, is it part of a family of related proteins, and if so, what are some of the names of the similar proteins? b) What is the metal involved? c) What is the coordination environment of the metal? d) What is known and generally accepted about the chemical role of the metal and its coordination environment in carrying out the function of the system? Pay attention to the experimental results presented in your Term Paper. What is learned about the system from the discussed experiment? In the conclusion, try to summarize what are some still unanswered questions that are motivating further research on the system that is the subject of your research topic. Prepare two potential questions to ask your colleagues during the discussion of your research topic, and be ready not only to ask but to answer them. 210 Exam Questions 1. Draw the electronic configurations of the given atom and ion and compare the relative stabilities of species. Indicate the number of unpaired electrons. 2. Draw the electronic configurations of a given element in the ground and possible excited states and predict its possible valence states. 3. Using the VB method, describe the structures of the given molecule and indicate the type of hybridization of the atoms. 4. Use the MO energy level diagrams to determine bond orders and multiplicities of the ground states of the fgiven molecules and ions and compare their relative stabilities. 5. Draw the MO energy level diagrams for the squaric (or tetrahedric, or hexahedtic) complexes of one of the transitional metal cation. Show the distribution of electrons in the field of a strong (or weak) ligand. 6. Comparing the relative stabilities of complex ions conclude on the possibility of their mutual transformations. 7. Describe the biological role of coordination compounds of Fe (or Zn, or Cu, or V, or Cr, or Mo, or Co) in biological processes. 8. Chelate effect in stabilisation of complexes by multidentate ligands. 9. Pearson’s concept of hard and soft interactions in coordination chemistry. 10. Protein structure. 11. Active sites of proteins. Coordination modes of amino acids. 12. A number of Copper (or Iron, or Zinc, or Cobalt) containing enzymes are known. Give the names of two of these and show the coordination environment around the metal. 13. The structures of iron centres of iron-sulphur proteins. 14. What are important properties of iron which can account for its extensive use in terrestrial biological reactions? 211 15. Aspects of storage (or transport, or biomineralization) of iron in the organisms. 16. Structure and characteristics of dioxygen-carrier proteins. 17. Electron structures of iron in deoxygenated and oxygenated forms of hemoglobin. 18. Other than hemoglobin oxygen-binding proteins. 19. Requirements for Effective Oxygen Carriers. 20. Cooperative and non-cooperative binding of dioxygen. 21. Wigner's spin selection rule. 22. Dioxygen reactions. 23. Roles of metalloenzymes in dioxygen reactions. 24. General approach to dinitrogen biofixation. 25. The role of Mo- (or V, or all iron) nitrogenases in the process of the dinitrogen fixation. 26. The role of quantum chemical modeling in bioinorganic chemistry. 27. Theoretical approach to determination of ionization constants of amino acids by potentiometric titration. 28. Calculation of stability constants of metal complexes with weak acids by potentiometric titration. 29. Theoretical approach to determination of ionization constants (deprotonation or protonation) of weak organic acids and bases by spectrophotometric titration. 30. Calculation of stability constants of metal complexes with organic ligands by spectrophotometric titration. 212 List of citations 1. Ivano Bertini, Harry B. Gray. Bioinorganic Chemistry, Mill Valley, California, 1994. 2. Glossary of terms used in Bioinorganic Chemistry. Ed. M. W. G. de Bolster. Pure &App. Chem., Vol. 69, No. 6, pp. 1251-1303, 1997. 3. Lorenz Kienle. Basics of Bioinorganic Chemistry. MaxPlanck-Institut für Festkörperforschung. Stuttgart. 4. S.J. Lippard, J.M. Berg, Principles of Bioinorganic Chemistry, U.S.B., 1994. 5. Bertini, I.; Gray, H. B.; Stiefel, E. I.; Valentine, J. S., editors. Biological Inorganic Chemistry. University Science Books, 2007. 6. Housecroft, C.E. and Sharpe, A.G. “Inorganic Chemistry, Pearson / Prentice Hall”, 2005 (2nd Edition). 7. Roat-Malone, R.M. Bioinorganic Chemistry: A Short Course. Wiley, 2002. 8. Kazuya Arashiba, Yoshihiro Miyake Yoshiaki Nishibayashi A molybdenum complex bearing PNP-type pincer ligands leads to the catalytic reduction of dinitrogen into ammonia Nature Chemistry, Vol. 3, 2011, pp. 120–125. 9. Jameson G.B. and Ibers J.A. Biological and Synthetic Dioxygen Carriers. http://authors.library.caltech.edu/25052/5/BioinCh_chapter4. pdf 10. Casiday R. and Frey R. Hemoglobin and the Heme Group: Metal Complexes in the Blood for Oxygen Transport. http://www.chemistry.wustl.edu/~edudev/LabTutorials/Hem oglobin/MetalComplexinBlood.html 11. http://simple.wikipedia.org/wiki/ 12. http://sandbian.wordpress.com/articles-papers/ 213 13. http://www.adichemistry.com/inorganic/bioinorganic/bioinor ganic-chemistry.html 14. Billo, E.J. (1997). "22". Excel for chemists: a comprehensive guide (2nd ed.). Wiley-VCH. ISBN 0-47118896-4. (Electronic) http://www.ahut.edu.cn/yxsz/ahkl/Teaching/Excel%20for%20 Chemists/Ch22.pdf. 15. A. Albert und E. P. Serjeant: Ionization Constants of Acids and Bases. 1. Auflage. Methuen & Co., London, John Wiley Sons, New York 1962. 179. 16. Beck, M.T.; Nagypál, I. (1990). Chemistry of Complex Equilibria. Horwood. ISBN 0-85312-143-5.Chapter 1. 17. Rossotti, F.J.C.; Rossotti, H. (1961). The Determination of Stability Constants. McGraw–Hill. 18. Koji Nakanishi, Philippa H. Solomon. Infrared Absorption Spectroscopy. 19. Silverstein, R.M.; Bassler, G.C.; and Morrill, T.C. Spectrometric Identification of Organic Compounds. 4th ed. New York: John Wiley and Sons, 1981. 20. http://facstaff.bloomu.edu/jmorgan/amino%20acid%20det ermination%20Spr09.pdf 21. http://orgchem.colorado.edu/Spectroscopy/irtutor/tutorial. html 22. http://www.chem.ucla.edu/~webspectra/irintro.html 23. http://en.wikipedia.org/ 24. http://www2.ups.edu/faculty/hanson/Spectroscopy/IR/IRfr equencies.html 214