* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download [Fe 4 S 4 Cys 4 ] 1
Survey
Document related concepts
Cooperative binding wikipedia , lookup
Protein folding wikipedia , lookup
Protein domain wikipedia , lookup
Protein structure prediction wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Circular dichroism wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein purification wikipedia , lookup
Protein moonlighting wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
List of types of proteins wikipedia , lookup
Western blot wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Transcript
Lecture 9 Redox metallo-biochemistry (continued) e- transfer proteins Cytochromes Fe-S proteins Blue copper proteins Kinetics of electron transfer reactions • Electron transfer between 2 metal centers can be either inner-sphere (via a bridging ligand) or outer-sphere (no bridging ligand, coordination spheres remain the same for both metal ions) • Only outer-sphere known for metalloproteins • Reasonably fast (> 10 s-1) over large distances (up to 30 Å) • Can be rationalised by Marcus Theory • Qualitatively: e- transfer is fast if the states before and after the redox reactions are similar (reorganisation energy is small) Cytochromes • Name comes from the fact that they are coloured • Differ by axial ligands and whether covalently bound • Involved in electron transfer (a,b,c) or oxygen activation (P450) • Essential for many redox reactions UV-Vis Spectra of cytochromes • classified by a bands: • a: 580-590 nm • b: 550-560 nm • c: 548-552 nm • (there’s also d and f) • all involved in electron transfer, all CN6 • P450: 450 nm: • Oxygen activation; CN5 Absorption spectra of oxidized (Fe(III) and reduced (Fe(II)) horse cytochrome c. Cytochrome c • Small soluble proteins (ca. 12 kDa) • Near inner membrane of mitochondria • Transfers electrons between 2 membrane proteins ( for respiration) • Heme is covalently linked to protein via vinyl groups (thioether bonds horse heart cytochrome c Bushnell, G.W., Louie, G.V., Brayer, with Cys) G.D. J.Mol.Biol. v214 pp.585-595 , 1990 • 1 Met and 1 His ligand •Conserved from bacteria to (axial) Man Cytochromes b • Heme has no covalent link to protein • Two axial His ligands • Shown is only soluble domain; the intact protein is bound to membrane F Arnesano, L Banci, I Bertini, IC Felli: The solution structure of oxidized rat microsomal cytochrome b5. Biochemistry (1998) 37, 173-84. Not for electron transfer: the cytochromes P450 • CN5, axial ligand is a Cys • 6th site for substrate/oxygen binding • Hydroxylates camphor P450Cam Tuning of heme function • In (deoxy)hemoglobin, Fe(II) is 5-coordinate • Must avoid oxidation to Fe(III) (Met-hemoglobin) • Neutral His ligand: His-Fe(II)-porphyrin is uncharged: Favourable • P450: Catalyses hydroxylation of hydrophobic substrates. Also 5-coordinate • 1 axial Cys thiolate ligand (negatively charged): Resting state is Fe(III), also uncharged • In cytochromes, CN=6: No binding of additional ligand, but very effective 1 e- transfer Iron-sulfur proteins Fe-S proteins • • • • Probably amongst the first enzymes Generally, Fe, Cys thiolate and sulfide Main function: fast e- transfer At least 13 Fe-S clusters in mitochondrial respiration chain • Rubredoxins: mononuclear FeCys4 site • Ferredoxins: 2,3 or 4 irons • Other functions: Aconitase: An isomerase IRE-BP: An iron sensor (see lecture 5) Rubredoxins: FeCys4 X-ray Structure of RUBREDOXIN from Desulfovibrio gigas at 1.4 A resolution. FREY, M., SIEKER, L.C., PAYAN, F. 1rfs: Spinach Fe2S2(Cys-S)4 1 awd: CHLORELLA FUSCA Fe2S2(Cys-S)2-(His-N)2: Rieske proteins Fe3S4(Cys-S)4 Fe4S4(Cys-S)4 1fda: Azotobacter vinelandii Fe-S clusters can be easily synthesised from Fe(III), sulfide and organic thiols, but are prone to rapid oxidation Richard Holm Self-assembly of Fe-S clusters Delocalisation of electrons: Mixed valence localized Fe3+ (red) and localized Fe2+ (blue) sites, and delocalized Fe2.5+Fe2.5+ pairs (green) Why e- transfer is fast: • Clusters can delocalize the “added” electron • minimizes bond length changes • decreases reorganization energy Fe-S proteins often contain more than one cluster: Azotobacter vinelandii: 2 clusters The five Fe-S clusters of the Fe-only hydrogenase from Clostridium pasteurianum • Activation of H2 • Active site (binuclear Fe cluster) on top • The other five Fe-S clusters provide longrange electron transfer pathways Pdb 1feh P cluster of nitrogenase FeMoCo cofactor cluster of nitrogenase Nitrogenase (Klebsiella pneumoniae) • Catalyses nitrogen fixation •N2 + 8H+ + 8e- + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi Redox potentials Tuning of redox potentials • For both heme proteins and Fe-S clusters, ligands coarsely tune redox potential • In [4Fe-4S] clusters, proteins can stabilise a particular redox couple • Further effects (a) solvent exposure of the cluster (b) specific hydrogen bonding networks especially NH-S bonds (c) the proximity and orientation of protein backbone and side chain dipoles (d) the proximity of charged residues to the cluster Tuning of redox potentials • Bacterial ferredoxins and HiPIPs: Both have Fe4S4Cys4 clusters • -400 mV vs. +350 mV • Ferredoxins: [Fe4S4Cys4]3- → [Fe4S4Cys4]2• HiPIPs: [Fe4S4Cys4]2- → [Fe4S4Cys4]1• HiPIPs are more hydrophobic: Favours -1 • NH...S bonds: 8-9 in Fd, only 5 in HiPIPs • Compensate charge on cluster; -3 favoured *) HiPIP: high potential iron-sulfur proteins Copper proteins Copper proteins • Oxidases • Cytochrome oxidase(s) • Enzymes dealing with oxides of nitrogen • Blue copper proteins • Superoxide dismutase • Tyrosinase • Caeruloplasmin Principles • Cu(II) forms the strongest M(II) complexes (see Irving Williams series) • Cu(I) also forms stable complexes • The Cu(I)/Cu(II) redox couple: 0.2V-0.8V • Most Cu proteins either extracellular or membrane-bound • Many Cu proteins involved in electron transfer Preferred geometries • Cu(II): Tetrahedron • Cu(I): trigonal planar or 2-coordinate Blue copper proteins • Azurin, stellacyanin, plastocyanin • Unusual coordination geometry: Another example for how proteins tune metal properties • Consequences: – Curious absorption and EPR spectra – High redox potential (Cu(I) favoured) • No geometric rearrangement for redox reaction: Very fast Blue copper proteins: coordination geometry 2.11 Å 2.9 Å Angles also deviate strongly from ideal tetrahedron (84-136°) Amicyanin (pdb 1aac) from Paracoccus denitrificans Key points • Properties such as redox potentials are tuned by proteins • Coarse tuning by metal ligands • Charge imposed by ligand can favour particular oxidation state • Geometry can be imposed by protein: Can favour particular oxidation state, and also increase reaction rate • Fine tuning by “second shell”: hydrophobicity, hydrogen bonds, charges in vicinity