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This paper is published as part of a Dalton Transactions themed issue on: Bioorganometallic Chemistry Guest Editor Charles Riordan University of Delaware, USA Published in issue 22, 2009 of Dalton Transactions Images reproduced with permission of John Peters (left) and Toshikazu Hirao (right) Papers published in this issue include: Perspective: Hydrogenase cluster biosynthesis: organometallic chemistry nature's way Shawn E. McGlynn, David W. Mulder, Eric M. Shepard, Joan B. Broderick and John W. Peters Dalton Trans., 2009, 4274, DOI: 10.1039/b821432h Role of aromatic substituents on the antiproliferative effects of diphenyl ferrocenyl butene compounds Ouardia Zekri, Elizabeth A. Hillard, Siden Top, Anne Vessières, Pascal Pigeon, Marie-Aude Plamont, Michel Huché, Sultana Boutamine, Michael J. McGlinchey, Helge Müller-Bunz and Gérard Jaouen, Dalton Trans., 2009, 4318, DOI: 10.1039/b819812h Resin-bound models of the [FeFe]-hydrogenase enzyme active site and studies of their reactivity Kayla N. Green, Jennifer L. Hess, Christine M. Thomas and Marcetta Y. Darensbourg Dalton Trans., 2009, 4344, DOI: 10.1039/b823152d Molecular structures of protonated and mercurated derivatives of thimerosal Wesley Sattler, Kevin Yurkerwich and Gerard Parkin Dalton Trans., 2009, 4327, DOI: 10.1039/b823467a Critical aspects of [NiFe]hydrogenase ligand composition Koji Ichikawa, Takahiro Matsumoto and Seiji Ogo Dalton Trans., 2009, 4304, DOI: 10.1039/b819395a Visit the Dalton Transactions website for more cutting-edge inorganic and organometallic research www.rsc.org/dalton and for more bioinorganic and biological inorganic chemistry papers in Dalton Transactions: www.rsc.org/Publishing/Journals/dt/News/BIB.asp Dalton Transactions An international journal of inorganic chemistry www.rsc.org/dalton Number 22 | 14 June 2009 | Pages 4261–4384 Themed issue: Bioorganometallic chemistry ISSN 1477-9226 PERSPECTIVE Peters et al. Hydrogenase cluster biosynthesis: organometallic chemistry nature’s way HOT ARTICLE Schatzschneider et al. Sonogashira and “Click” reactions for the N-terminal and side-chain functionalization of peptides with CO releasing molecules 1477-9226(2009)22;1-2 www.rsc.org/dalton | Dalton Transactions PERSPECTIVE Hydrogenase cluster biosynthesis: organometallic chemistry nature’s way Shawn E. McGlynn, David W. Mulder, Eric M. Shepard, Joan B. Broderick and John W. Peters* Received 1st December 2008, Accepted 11th March 2009 First published as an Advance Article on the web 30th March 2009 DOI: 10.1039/b821432h It has been over a decade now since it was revealed that the metal containing active sites of hydrogenases possess carbonyl and cyanide ligands bound to iron. The presence of these ligands in hydrogenases came as a surprise and to-date these ligands have not been observed to be associated with any other enzymatic metallocenter. The elucidation of the structures of these unique metalloenzymes and their associated metal clusters created opportunity for a number of different lines of research. For synthetic chemists, the structures of hydrogenase active sites have provided attractive targets for syntheses that advance our understanding of the electronic structure and reactivity of these unique enzyme active sites. These efforts contribute to the synthesis of first row transition metal catalysts for hydrogen oxidation and hydrogen production that could have significant impacts on alternative and renewable energy solutions. Although effective synthetic approaches have been identified to generate models with a high degree of similarity to these active sites, the details of how these metal clusters are synthesized biochemically have not been resolved. Since hydrogen metabolism is presumed to be an early feature in the energetics of life and hydrogen metabolizing organisms can be traced very early in molecular phylogeny, the metal clusters at hydrogenase active sites are presumed to be among the earliest of known co-factors. Comparison of mineral based precursors and synthetic cluster analog chemistry to what is observed in contemporary biological systems may shed light on how proto-metabolically relevant catalysts first arose prebiotically by the processes of adoption of pre-existing functionality and ligand assisted catalysis. Introduction Hydrogenases occur in three forms [FeFe]-, [NiFe]-, and [Fe]hydrogenase so named to reflect their respective metal cluster content. The [FeFe]- and [NiFe]-hydrogenases formally catalyze reversible interconversion between dihydrogen gas and protons and electrons where the [Fe]-hydrogenases which occur exclusively Department of Chemistry and Biochemistry and the Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, Montana 59715, USA. E-mail: [email protected]; Fax: +1 406994-7470 in the methanogenic Archaea are given the systematic name, H2 -forming methylene-H4 MPT dehydrogenase (Hmd), and catalyze the dehydrogenation of methylene-tetrahydromethanopterin (methlylene-H4 MPT). Hydrogenases are widely distributed in microorganisms.1,2 Hydrogen oxidation is used to couple the generation of reducing equivalents to energy yielding reactions to support microbial growth, while hydrogen production is utilized as a mechanism to recycle reducing equivalents that accumulate during fermentative metabolism. In nature, hydrogen oxidizing and hydrogen producing microorganisms frequently occupy the same or adjacent David Mulder was born in 1983. He studied chemistry at Calvin College, receiving his B.S. degree in chemistry in 2005 and is currently a Ph.D. candidate in Prof. John Peters research group at Montana State University where he received an INBRE research fellowship. His scientific interests are in iron–sulfur enzyme biochemical and spectroscopic characterization and hydrogenase biosynthesis. Shawn McGlynn was born in 1983. He received a B.S. degree in chemistry from Montana State University in 2005 and is currently a Ph.D. candidate in Prof. John Peters research group at Montana State University and an IGERT fellow in Geobiological Systems. His research interests include metal usage in biology, hydrogenase active site assembly, and the origins of life. Shawn E. McGlynn 4274 | Dalton Trans., 2009, 4274–4285 David W. Mulder This journal is © The Royal Society of Chemistry 2009 environmental niches and interspecies hydrogen transfer is of paramount importance to the energetics and equilibrium processes of microbial systems. So large is the role of hydrogen in microbial ecosystems that interspecies hydrogen transfer and the associated interdependence of hydrogen oxidizing and hydrogen producing organisms has been suggested in two independent hypotheses as having been the driving force of the evolution of the first eukaryotic cell.3,4 The occurrence of hydrogenases in microbial hosts is an interesting mystery of molecular evolution since to date [FeFe]hydrogenases have been found to be associated with bacteria and lower eukaryotes such as algae and protists, but are not found to be associated with archaea or cyanobacteria. In contrast, the [NiFe]-hydrogenases, which are found to be associated with a wide variety of bacteria, are also commonly found in many archaea and cyanobacteria but are not found to be associated with any lower eukaryotes. Finally, the occurrence of the [Fe]-hydrogenases is observed exclusively in some hydrogenotrophic methanogenic archaea. These discreet differences in the occurrence of the different classes of hydrogenases is surprising given the common features of their enzyme active site ligand architectures and Eric Shepard was born in 1977. He received a B.S. degree in chemistry from Rocky Mountain College. He studied copper and TPQ-containing amine oxidases under Dr David M. Dooley at Montana State University, where he was supported by a NSF IGERT fellowship on complex biological systems. He received his Ph.D. degree in biochemistry from MSU and is currently a postdoctoral research associate Eric M. Shepard under Dr. Joan B. Broderick. His research interests are metal cluster assembly in the [FeFe] hydrogenase system and [Fe–S] cluster spectroscopy and reactivity. Joan Broderick was born in 1965. She received a B.S. from Washington State University and a Ph.D. from Northwestern University. She was an American Cancer Society postdoctoral fellow at MIT before joining the faculty at Amherst College as Assistant Professor in 1993. She moved to Michigan State University in 1998 and to Montana State University in 2005, where she is currently Professor of Chemistry and Biochemistry. Her research Joan B. Broderick interests are in mechanistic bioinorganic chemistry, with a particular focus on enzymes utilizing iron–sulfur clusters to catalyze radical reactions. This journal is © The Royal Society of Chemistry 2009 indicate that these enzymes have evolved to common ligand architectures and reactivity by convergent evolution. Interest in hydrogen metabolism and hydrogenases has grown exponentially over the past decade in part due to the growing interest in alternative and renewable energy solutions. Hydrogen production by microorganisms or the use of hydrogenase enzymes or mimetic compounds as hydrogen oxidation or hydrogen production catalysts has tremendous promise; however there are significant barriers to surmount. Overcoming these barriers will require an intimate understanding of the biosynthesis and maturation of hydrogenase enzymes as well as their structure and reactivity. As thoughts concerning hydrogenase structure, function, and reactivity continue to evolve, the relationships between the features of hydrogenase active sites and those of other complex iron-sulfur enzymes and iron-sulfur mineral structures and reactivity have also been gaining interest. Given that complex iron-sulfur enzymes are involved with reactions such as reversible hydrogen oxidation, nitrogen fixation, and reversible carbon monoxide oxidation— reactions that would have been important in the prebiotic earth and necessary for the first stages of the emergence of life—it is rational to think about these enzymes as having an ancient origin. It is possible that the unique metal-containing prosthetic groups at the active sites of these enzymes may represent the oldest of known co-factors with what is observed today existing as highly evolved versions of modified iron-sulfur mineral catalysts. Investigations of hydrogenase structure, function, and biosynthesis may in fact provide some keys as to the origin of life and perhaps even contribute to the development of signatures for life beyond Earth. Given the potential importance of hydrogenases at the origin of life and the potential for hydrogenase based catalysis for renewable energy and sustaining life on Earth, hydrogenase research is an ideal combination of basic and applied research. Active site metal cluster structure In 1995, the first structure of a [NiFe]-hydrogenase was determined by X-ray diffraction methods from the sulfate reducing bacteria (SRB) Desulfovibrio gigas5 and with the structure came some John Peters was born in 1965. He received a B.S. in Microbiology from the University of Oklahoma and a Ph.D. from Virginia Tech. He was a NIH postdoctoral fellow at Cal Tech before joining Utah Sate University as Assistant Professor in 1997 where he was the recipient of the Camille Dreyfus Teacher-Scholar Award. In 2002, he joined Montana State University and is currently Professor of Chemistry and Biochemistry and Director of the NASA John W. Peters funded Astrobiology Biogeocatalysis Research Center and the MSU Thermal Biology Institute. His research interests are in complex iron-sulfur enzymes, specifically hydrogenase and nitrogenase structure, function and biosynthesis. Dalton Trans., 2009, 4274–4285 | 4275 surprises (Fig. 1a). From this work, it was revealed that the active site possessed an Fe atom that was previously missed by biochemical and spectroscopic work. Even more intriguing was that the Fe was coordinated by a number of non-protein ligands that could not be resolved by X-ray crystallography. The nonprotein ligands were determined to be cyanide and carbonyl ligands by Fourier transform infrared (FTIR) spectroscopy and chemical analysis, which resolved long standing issues concerning the interpretation of carbon monoxide inhibited states of the hydrogenase.6 Through this work, the active site of the D. gigas [NiFe]-hydrogenase was found to exist as a heterometallic cluster with a Ni ion coordinated by four cysteine thiolates to an Fe ion by two bridging thiolates. Additional coordination to Fe is supplied by three diatomic ligands (two cyanide and one carbonyl) and a bridging species for which its composition has been proposed to be a m-oxo, sulfido, hydroxo, peroxide, or sulfoxide group7–9 (Fig. 1b). Structural characterization of the [FeFe]-hydrogenase came later from Clostridium pasteurianum (CpI) and Desulfovibrio desulfuricans10,11 (Fig. 1c) and revealed a similar iron coordination environment yet a somewhat different overall active site composition. Common to these hydrogenases are the presence of several accessory iron–sulfur clusters, which presumably function to shuttle electrons to and/or from external physiological electron donors and/or acceptors. For the [FeFe]-hydrogenase from Clostridium pasteurianum the apparent bifurcated pathway with two separate accessory clusters approaching the surface at different positions may suggest that the enzyme can accommodate alternative electron donors and/or acceptors. The six iron catalytic “H-cluster” active site (Fig. 1d) is comprised of a [4Fe-4S] cubane bridged via a single cysteinyl thiolate to a 2Fe subcluster in which both irons are coordinated by a CO and CN- ligand, and bridged by a m-CO ligand and a non-protein dithiolate of still unknown composition having been proposed to exist as propane dithiolate, dithiomethylamine12 , or dithiomethylether.13 Knowledge as to the stereochemical position of the CO and CNligands on the active site was based on CO and CN- ability for hydrogen bonding.11 Subsequent FTIR studies revealed that these enzymes are reversibly inhibited by CO12,14 and the structure of the CO-inhibited enzyme was determined via X-ray crystallography from crystals of the C. pasteurianum enzyme grown in the presence of CO.14 The structure revealed reversible hydrogen oxidation likely occurs at a ligand-exchangeable site at the distal iron atom of the 2Fe subcluster in relation to the [4Fe-4S] cluster. In the presumed oxidized state of the C. pasteurianum enzyme, a water molecule occupies the ligand-exchangeable site. However, this water molecule is not present in the D. desulfuricans [FeFe]hydrogenase structure as it is believed to represent a more reduced state of the active site. The [Fe]-hydrogenases were first shown to contain iron ligated by CO ligands in 200415 and very recently, the structural description of the [Fe]-hydrogenase was revealed with bound cofactor allowing for the first time side by side comparison of these unique active sites.16 The [Fe]-hydrogenase active site exists with iron coordinated by two CO ligands, a cysteinyl sulfur, the Fig. 1 Ribbon/space filling diagram of the (A) [NiFe]-hydrogenase from D. gigas (PDB code 1YQ98 ). (B) [FeFe]-hydrogenase from C. pasteurianum (PDB code 3C8Y13 ). For both structures, the protein domains are represented with different colors. For D. gigas, a small and a large subunit exist, for which they can be divided into two and five separate domains, respectively, according to protein folds. Atomic models of the [NiFe]- and [FeFe]-hydrogenase active site metal clusters are shown for each (B and D), respectively, (Fe: dark red, S: orange, O: red, N: blue, C: dark grey, unknown atom of dithiolate ligand: magenta). For the D. gigas model, a bridging peroxide ligand is shown and represents the oxidized “unready” state of the active site. The C. pasteurianum active site depicted is presumed to represent the oxidized form as a water molecule is present at the ligand-exchangeable site of the distal Fe atom. All ribbon diagrams and atomic models were generated in PyMOL.103 4276 | Dalton Trans., 2009, 4274–4285 This journal is © The Royal Society of Chemistry 2009 nitrogen of a 2-pyridinol compound, and a ligand of unknown composition. Collectively, these enzyme active sites and features presumably allow for the stabilization of reduced iron in a form capable of catalyzing the hydrogenase reaction, and in the case of the [Fe]hydrogenase center, the dehydrogenation of methylene-H4 MPT. The nature of the evolutionary and biochemical origins of these complexes, as well as the electronic and structural basis for their reactivity, poses an array of intriguing questions, which are being addressed by research groups from a diverse range of backgrounds and expertise. Active site cluster mimics The syntheses of hydrogenase active site analogs began soon after the first structural report of the [NiFe]-hydrogenase when workers were motivated by the prospects of creating biomimetic catalysts and gaining further insight into the reactivity of the hydrogenase active site clusters. Significant progress has been made in generating active site cluster mimics for both [NiFe]- and [FeFe]-hydrogenases and this work has been the topic of recent reviews.17–20 A more topical summary of the progress in the field is described here. The development of synthetic techniques suitable for the formation of heterobimetallic sulfur bridged CO and CN- ligated clusters posed unique challenges to the field of organometallic chemistry, and in the case of the [NiFe] center, the events leading to the generation of sulfur coordinated Ni species ligated in bidentate fashion to iron coordinated by CO and CN- were major highlights. Within a year of the first structural description of the [NiFe]hydrogenase, Darensbourg and co-workers21 reported a heterobimetallic structural analogue (Fig. 2a) obtained by the reaction of a sulfur coordinated Ni2+ complex with an iron carbonyl. The resultant structure consisted of a nickel moiety bound to a Fe(CO)4 fragment via a single bridging sulfur. In 1997, a [NiFe]hydrogenases active site model compound in which nickel was linked to iron by two sulfur atoms (Fig. 2b) was reported22 from the reaction of an Fe(CO)2 (NO)2 with a nickel complex to give the formation of a heterometallic [NiFe] complex with Fe ligated by NO. The formation of mimics with heterometallic [NiFe] complexes coordinated to CO ligated Fe came later with the complex synthesized by the reaction of an iron thiolate with NiCl2 (dppe) under an atmosphere of CO23 (Fig. 2c). In 2002, the formation of an analog with Ni ligated solely by thiolates was achieved24 (Fig. 2d), marking a significant advance from other syntheses, which utilized phosphines as soft donor ligands.23,25 An additional advancement in bimetallic heteroatomic [NiFe] analogue synthesis came in 2005; when it was realized that reaction of the pre-formed iron carbonyl [Fe(CO)3 (CN)2 Br]- with a nickel complex gave a fully sulfur coordinated Ni moiety bridged to iron ligated by two CO and two CN- molecules26 (Fig. 2e). This development represented the culmination of many years of work by a diverse group of chemists to develop the reaction specificity to yield organometallic compounds with three main qualities requisite for [NiFe]-hydrogenase active site resemblance: sulfur coordinated Ni bound via two sulfur thiolates to an iron center ligated by both CO and CN- . Synthetic efforts to mimic the [NiFe] site continue to advance the understanding of H2 activation by the active site, with a recent report suggesting that coordination of This journal is © The Royal Society of Chemistry 2009 Fig. 2 Selected synthetic structures mimicking the active sites of the (left) [NiFe]-hydrogenase active site and (right) [FeFe]-hydrogenase active site (discussed in text). hydrogen by the [NiFe] center stems from an ability of Ni in the active site to shift from square pyramidal to octahedral geometry, this being based upon observations of various [NiFe] complexes.27 In the midst of efforts to develop [NiFe] active site models, structural descriptions of the [FeFe]-hydrogenases in 1998 and 199910,11 catalyzed the development of a parallel vein of research aimed at mimicking the related but distinct different active site structures. Similar challenges were faced in the case of [FeFe]hydrogenase active site analog synthesis as in the case of the [NiFe]-hydrogenases but with some variations. Beginning work was directed at the synthesis of the 2Fe-subcluster of the H-cluster. The previous synthesis and characterization of various diiron carbonyl complexes, dating back to 192828,29 (Fig. 2f) and furthered in the 1980’s, provided a starting point for the synthesis of H-cluster mimics and thus allowed the community to hit the ground running. These syntheses demonstrated the formation of the di-ironhexacarbonyldisulfide compound [Fe2 (SCH2 CH2 CH2 S)(CO)6 ] present with a propane dithiolate30–32 starting from either Fe3 (CO)12 or (m-S2 )Fe2 (CO)6 (Fig. 2g left). Following structural elucidation of the [FeFe]-hydrogenase, two of the CO ligands on this compound were substituted with CN- by three different groups33–35 via reaction of Fe2 (pdt)(CO)6 (pdt = propyl dithiolate) with two equivalents of CN- (Fig. 2g right) to give a remarkable analog of the H-cluster 2Fe-subcluster exhibiting a short Fe-Fe distance (2.6 Å), and the presence of both CO and CN- ligands. The parent compound for this reaction was again a remnant of non-intentional hydrogenase active site mimicry carried out some thirty years prior to the knowledge of Dalton Trans., 2009, 4274–4285 | 4277 the active site structure; King showed in 196136,37 the development of chemistry suitable for the thiolation of iron carbonyls by reaction with alkyl sulfides, allowing m-sulfur-iron bond formation of di-iron centers. Subsequent work on the di-iron alkyl-sulfide ligated compounds resulted in variations in which the dithiolate linkage was varied with central atomic positions being occupied by ether38,39 and amine40 (Fig. 2h) functional groups, as opposed to the methylene group at this position in the initial propane dithiol ligated compounds. These variations were elicited by the inability to assign the central atom of the bridging dithiolate based on crystallographic data since the likely possibilities (CH2 , O, NH, NH2 + ) are isoelectronic. A further challenge presented in the development of structural analogs included adding the presence of a m-CO ligand. This goal was first met by Pickett and co-workers41 with the formation of a mixed valent Fe1+ Fe2+ compound with a m-CO ligand, although this compound proved to be very unstable. Later work by two groups gave stable m-CO containing di-iron compounds42,43 (Fig. 2i) and in addition to these CO bridged compounds, a model with a bridging hydride capable of binding and activating H2 at an Fe2+ site has been reported.44,45 However, these compounds did not have CN- ligands and instead were coordinated by bulky IMES, PMe3 , and dppv ligands, respectively, which presumably functioned to stabilize the unique geometry that the m-CO ligand demands. A major advancement in the synthesis of H-cluster mimics came in 2005 with the synthesis of the complete 6Fe framework of the H-cluster46 (Fig. 2j). The bridging of a di-iron carbonyl subcluster to a [4Fe-4S] cubane was accomplished by first capping Fe3 (CO)12 by reaction with a three sulfur containing thioester with the subsequent product being reacted with a thiolate coordinated [4Fe-4S] cubane. This general approach allows significant flexibility for the formation of additional analogue targets as well as providing a means to probing cluster characteristics as has been recently demonstrated for the investigation of CO binding at a 2Fe center.47 Mimetic synthesis of hydrogenase active sites metal clusters resulted in the development of a suite of chemical approaches. From a synthetic perspective, the starting materials for a number of these compounds are simple iron carbonyl complexes such as Fe(CO)5 , Fe2 (CO)9 , and Fe3 (CO)12 , to which sulfur in the form of sulfide or thiolates and CN- from compounds such as Et4 NCN, and a variety of nickel complexes may be added. In terms of catalysts for hydrogen energy technologies, platinum chemistry is very effective at catalyzing reversible hydrogen oxidation at little or no overpotential, however, with the limited availability of platinum there is a real need for examining non-noble metal based solutions to accomplishing this chemistry. Lessons learned in synthetic efforts are thus valuable for the continued pursuit of hydrogenase active site mimics that may well yet prove to be catalytic solutions for the accumulation of H2 as a fuel currency, as well as providing a vantage point from which to consider the biochemical events that led to the formation of these interesting catalytic co-factors. Biosynthesis and maturation Biological utilization of metal-sulfide protein bound co-factors, which are ubiquitous in living systems, stems from the inherent electronic and reactive properties of both iron and sulfur.48 4278 | Dalton Trans., 2009, 4274–4285 The utilization and assembly of these co-factors necessitates the operation of a complex and specialized set of proteins to allow for the coordinated and regulated delivery of atoms in a way that avoids the inherent toxicity of free aqueous metal ions.49–52 The formation of the hydrogenases provides additional biosynthetic challenges and has long puzzled biochemists as to the molecular mechanisms responsible for the harnessing of the unique ligand set of the active sites. The collective features of the hydrogenase co-factors require the operation of a specialized set of proteins to biosynthesize the active site cluster. Given the similarity in overall composition between the hydrogenase active sites, it is interesting if not surprising that the generation of the hydrogenase active sites appears to occur by non-redundant chemistry, with distinct sets of maturation proteins being required for active site synthesis of each of the hydrogenases. A summary of the proposed activities for gene products involved in [NiFe]- and [FeFe]-hydrogenase active site cluster biosynthetic pathways is provided in (Fig. 3) and is discussed further below. [NiFe]-hydrogenase active site synthesis has been studied in a number of organisms including Ralstonia eutropha, Bradyrhizobium japonicum, Rhizobium leguminosarum, Azotobacter species, and E. coli and proceeds by the action of at least six accessory proteins, with seven being required in cases in which proteolytic processing is required at the final step of maturation.53 The proposed roles of individual proteins in the maturation process for E. coli hydrogenase 3 is summarized in (Fig. 4A) and begins with the generation of cyanide from carbamoylphosphate by the action of the two proteins HypF and HypE. In a two step process that requires two ATP hydrolyzing events, HypF transfers the carboxamide group of carbamoylphosphate to HypE as thiocarboxyamide where dehydration occurs to yield an enzyme bound thiocyanate, which can later be donated to iron.54–57 Subsequent interaction of thiocyanate bound HypE with the HypD/HypC complex results in the donation of the CN- ligand to an iron bound to HypD/HypC.58 The CN- ligated iron bound by the HypD/HypC complex is then transferred by HypC to the large subunit of the hydrogenase enzyme.54 Still in question is the metabolic source of the CO ligand and at which stage it is incorporated. Carbamoylphosphate has been ruled out as the source of CO by labelling studies59,60 and the utilization of a pathway involving acetate has been suggested.61 With CO and CNcoordinated Fe now present on the large subunit, Ni is inserted by the actions of the Ni binding GTPase HypB62,63 and the Ni binding HypA.64 The final step in [NiFe]-hydrogenase maturation involves proteolytic cleavage of a C-terminal extension, if present. [FeFe]-hydrogenase active site biosynthesis shares many of the chemical challenges as just outlined for the [NiFe]-hydrogenase site, but from what is known thus far appears to require fewer gene products perhaps due to the presence of only a single type of metal ion in the active site1 . Indeed, [FeFe]-hydrogenase maturation was found to be dependent on three gene products (HydE, HydF and HydG) based on analysis of deletion mutants of Chlamydomonas reinhardtii in which the gene products HydEF and HydG were identified to be required for the accumulation of active hydrogenase.65 Subsequent analysis revealed that the gene products HydE, HydF, and HydG are conserved in organisms harbouring an active [FeFe]-hydrogenase, with HydEF existing as a fusion product in C. reinhardtii. Based on sequence comparison, the two proteins HydE and HydG were predicted to belong to the This journal is © The Royal Society of Chemistry 2009 Fig. 3 Overview of genes involved in the biosynthesis of the [NiFe]-hydrogenase and [FeFe]-hydrogenase active site and their proposed functions. radical S-adenosylmethionine (AdoMet) family of enzymes, and HydF was predicted to bind an iron-sulfur cluster in addition to having the ability to hydrolyse nucleotide tri-phosphates.65 Following this discovery, an E. coli based heterologous expression allowing the co-expression of the [FeFe]-hydrogenase structural protein with the three accessory proteins was developed and shown to be sufficient for heterologous maturation, paving the way for future biochemical studies. In subsequent work, the gene based predictions for the enzymatic activities of these enzymes was corroborated by analysis of anaerobically reconstituted Hyd accessory protein homologues from the organism T. maritima.66, 67 The AdoMet class of enzymes comprises a large family of ironsulfur cluster containing enzymes capable of catalyzing a wide range of free radical initiated reactions including carbon-carbon bond formation, isomerisations, and sulfur insertions68 ; thus a number of chemistries may be envisaged as taking part in H-cluster biosynthesis. Knowledge of the enzyme families to which the [FeFe]-hydrogenase assembly factors belong to led to a hypothesis based off pre-established reactivity of AdoMet enzymes in which the activities of HydE and HydG, respectively, are proposed to act to generate the CO and CN- ligands, as well as the bridging dithiolate ligand.69 The three maturation proteins are presumed to be responsible in total for the generation of the ligand set of the [FeFe] subcluster with the possibility that CO could be generated through the action of carbon monoxide dehydrogenase excluded as [FeFe]-hydrogenases are found in organisms which do not contain this enzyme. In our hypothesis for [FeFe]-hydrogenase maturation, the three gene products HydE, HydF, and HydG are thought to be directed solely at the synthesis of the 2Fe subunit of the [FeFe] active site, as the [4Fe-4S] moiety presumably would not require the action of a specific set of assembly proteins but instead be formed via the action of the endogenous host iron-sulfur cluster assembly machinery. HydE or HydG is envisioned as acting to generate the bridging dithiolate linkage in a mechanism analogous to that of the radical-AdoMet protein LipA, which catalyzes carbon– This journal is © The Royal Society of Chemistry 2009 sulfur bond formation; such C–S bond formation, occurring at a bridging sulfide of an iron–sulfur cluster, would be expected to shift the reactivity from the sulfur atoms to the irons of the cluster. Following alkylation of the sulfides, a radical-initiated amino acid decomposition of glycine is proposed to occur by the action of HydE or HydG, resulting in the formation of CO and CN- . The first step of this latter proposed reaction is analogous to the chemistry catalyzed by the radical-AdoMet enzyme pyruvate formate-lyase activating enzyme, lysine amino mutase, or ThiH involved in thiamine biosynthesis which involves the generation of an amino acid radical intermediate. These ligand-forming events are hypothesized to result in assembly of the 2Fe subcluster of the H-cluster on one of the accessory proteins. At this stage the role of GTP binding and hydrolysis by HydF is unknown, however, our results concerning in vitro activation of the [FeFe]hydrogenase would suggest that these activities are not directed at cluster insertion as originally proposed. Definitive knowledge as to the roles of the accessory proteins in active site formation has been advancing rapidly, due in large part to development of an in vitro system for the activation of the structural hydrogenase protein.70 This in vitro system has led to the recent identification of HydF as a scaffold or carrier protein which acts at the terminal step of H-cluster biosynthesis71 , presumably delivering the 2Fe sub-cluster to the immature structural protein (Fig. 4B). Currently unresolved issues relate to the enzymatic roles and substrates of the two AdoMet proteins HydE and HydG, the precise nature of the cluster precursor bound by HydF prior to delivery, and the role of GTP hydrolysis by HydF in the maturation process. Very little is known regarding the biosynthetic route for the case of the [Fe]-hydrogenases. As is the case between the [NiFe]- and [FeFe]-hydrogenases, however, phylogenetic evidence indicates that the structural protein is evolutionarily isolated and stems from a separate lineage, suggesting a separate route for the generation of CO ligands at the iron center. One possible and Dalton Trans., 2009, 4274–4285 | 4279 Fig. 4 Overview of the metal cluster assembly processes for the (A) [NiFe]- and (B) [FeFe]-hydrogenases. The [NiFe]-hydrogenase maturation scheme is based on hydrogenase-3 in E. coli and the [FeFe]-hydrogenase maturation model is thought to be general to organisms containing this enzyme. (A) CN- is generated by the action of HypE and HypF utilizing carbamoylphosphate and ATP forming thiocyanate bound by HypE. The CN- group on HypE is transferred to the iron coordinating HypD/HypC complex, which is then transferred to the large subunit of the structural hydrogenase protein. The source of CO is unknown. HypA and HypB act to deliver nickel, and in most cases a C-terminal peptide of the hydrogenase is endopeptidically cleaved completing the maturation process. (B) The two SAM radical enzymes HydE and HydG are proposed to impose non-protein cluster ligands on an existing [2Fe-2S] rhomb in two separate steps: (i) the formation of the dithiolate linkage and (ii) CO and CN- ligands, possibly from an amino acid precursor on the scaffold protein HydF. HydF subsequently delivers the ligand modified 2Fe unit to the immature structural protein, which is presumed to exist with the presence of a preformed [4Fe-4S] cluster, accomplishing activation. 4280 | Dalton Trans., 2009, 4274–4285 This journal is © The Royal Society of Chemistry 2009 intriguing correlation does exist however, as the gene encoding a radical-AdoMet protein has been observed to cluster with the gene encoding the [Fe]-hydrogenase structural protein in a number of cases.72 Given this observation, it is interesting to speculate about the commonality of radical-AdoMet chemistry in the generation of essential CO ligands for both the [Fe]and the [FeFe]- hydrogenases and that the CO of both these hydrogenases may be formed through a radical initiated amino acid decomposition event. Despite the distinct differences in the maturation systems for the [NiFe]-, [FeFe]-, and [Fe]-hydrogenases, similar chemical and catalytic functionality can be found. In the case of the [NiFe]and [FeFe]-hydrogenases, a cluster precursor has been proposed to be assembled on a scaffold protein prior to insertion into the immature hydrogenase structural protein, and in both cases GTPase activity has been implicated in having a role in the maturation process. These two hydrogenase maturation systems differ however in their respective routes to the generation of the diatomic ligands CO and CN- . Insofar as organisms harbouring hydrogenases are able to occupy niches unavailable to organisms without these enzymes, the functional, catalytic, and maturation pathway similarities between hydrogenases attests to the ability of evolution to produce functionally similar yet distinct solutions to environmental problems. This points to an apparent optimum of hydrogenase catalytic activity being associated with CO and CNligated iron, presumably as a direct consequence of the ability of these ligands to stabilize metals in low oxidation states. In short, three distinct hydrogenase enzymatic systems have convergently evolved to contain, in whole or in part, iron coordinated by small inorganic p acid ligands (CO and/or CN- ), suggesting that the low valent iron stabilized by such p acids has been a central driving force in hydrogenase evolution. Prebiotic mineral clusters, ligand accelerated catalysis and ancient enzymes Hydrogen occupies a central role as a mobile electron carrier in biological systems. Given the wide diversity of organisms that utilize hydrogen oxidation/reduction in metabolic processes, as well as the potential to derive high concentrations of hydrogen geochemically via processes such as serpentinization73,74 and the high concentration of hydrogen present on the early earth75 , it is reasonable to suppose that hydrogen as a mobile electron carrier and potent reductant has been coupled to metabolic processes from their inception.2,74,76 The wide distribution of hydrogenases in organisms and the presence of hydrogen on the early earth thus make the consideration of hydrogenases and the events which led to the formation of hydrogen oxidation and reduction catalysts especially relevant to the consideration of early energetics and the origin of life. In addition to reversible hydrogen oxidation, small molecule interconversions and cycling of the major atomic constituents of life play a critical role in contemporary living systems and must have also participated in the processes accompanying the transitions from an abiotic to a biotic world. Given that processes including nitrogen fixation, reversible carbon monoxide oxidation, and hydrogenase activity occur biologically via the utilization of metal clusters, it is possible that these clusters in biological systems are signatures and even reflections of ancient metabolic This journal is © The Royal Society of Chemistry 2009 and prebiotic processes in which transition metal based catalysts occupied salient roles. Metal sulfides and metallic transition metals have been shown to function as catalysts for biologically relevant reactions and these catalytic activities may have been requisite for the accumulation of molecules later incorporated into living systems. Activities associated with metallic, metal sulfide, and metal oxide based catalysts include the reduction of N2 to NH3 77–79 , the synthesis of pyruvate80 and amino acids, and peptides.81,82 These activities strongly suggest that transition metals, which feature so prominently in contemporary biology, were relevant to chemistry associated with the development of life. As well as occupying an important role in individual reactions, a further important role of both prebiotic and contemporary catalysts is that of the reaction network connector. In the sense that catalysts bound the metabolic capacity and flexibility of an organism, the formation of such catalysts prebiotically may have been a major barrier in the origin of life. Catalysts in metabolic networks function to kinetically channel products and substrates at time scales allowing for sufficient interconnectivity to result in the ability to couple energy yielding reactions to those that are utilized by organisms to maintain function. With these activities in mind the importance of catalysis in the origins of biological complexity can perhaps not be overstated. Taking into account the aforementioned chemistry underlying the creation of hydrogenase active sites both biologically and synthetically results in a unique perspective from which to consider the origins of prebiotic catalysts. A greater understanding of (i) the extent to which minerals present on the early Earth may have harboured preexisting catalytic functionality, (ii) the ability of metal clusters to form and the role of this in the formation of biologically accessible molecules, and (iii) the contribution of ligand modifying events to the emergence of catalytic functionality, may together yield insight as to the nature or the original catalysts and catalytic events which led to the emergence of life. The present day biological utilization of the metal sulfide cofactors observed in hydrogenases and other enzymes to accomplish efficient reactivity hints at the possibility that these co-factors themselves may have been derived from geologically related mineral precursors and thus have been adopted, modified, and refined through evolutionary time to become the extant clusters observed today existing as products of geomimicry. Various ironsulfur minerals exhibit structures similar to those seen in biology, and attention has been drawn to the resemblance of biological clusters such as that observed in the enzyme CODH and that of the mineral greigite83 , as well as a similarity in geometry between the mineral mackinawite and the di-iron center of the [FeFe]hydrogenase (Fig. 5a).84 These “ready made” clusters have been proposed to have provided a structural basis for the catalytic events required in the origin of life84 , itself being a product at least in part of inherent geological functionalities. Along with the similarities existing between mineral and enzyme active site structure, an emerging body of work has contributed to the understanding of how ligand modifications, including carbonylation, can occur in prebiotic conditions. Iron carbonyl complexes, which as described above form the reactive starting material of many active site analogs, have been synthesized from ferrous iron upon the addition of CO2 85 , CO and ethane thiol86 , and as well as from aqueous preparations in a simulated hydrothermal reactor upon the reaction of iron sulfide with CO.80,87 Dalton Trans., 2009, 4274–4285 | 4281 Fig. 5 Possible functional roles of prebiotic metal sulfides existing as (A) “ready made” as described by Russell in which some metal sulfides exist with lattice structures of striking similarity to clusters observed in biology. Here, the iron sulfide mackinawite is shown, highlighting resemblance of the mineral lattice structure to the 2Fe unit of the [FeFe]-hydrogenase, albeit existing without ligand modifications (adapted from ref. 84). (B) Possible formation of the amino acid glycine via the condensation of iron bound CO and CN- with H2 O in a reverse reaction to that proposed to occur in the biosynthesis of CO and CN- from glycine. (C) An iron-sulfur mineral surface, shown to be capable of ligand-accelerated autocatalysis and feedback as described by89 as a means of catalyst formation and diversification. The mineral surface is initially capable of carrying out catalytic formation of a molecule that subsequently binds to the mineral surface, and via ligand accelerated catalysis enhances the formation of more of the same molecule. In this way, the catalyst is selected for kinetically, propagating itself through ligand-assisted autocatalysis. In addition to these carbonylated iron species, iron cyanide species in the form of Prussian blue have been isolated from Miller–Urey types of experiments showing that spark discharge experiments are sufficient for the generation of CN- , which reacts with ferrous iron.88 The observation of iron carbonyl and iron cyanide species indicate that the unique ligand set of the hydrogenase enzymes may be accessible in prebiotic conditions. Furthermore, iron cyanide synthesis has been shown to be feasible with nucleophilic iron and electrophilic CN- and in the reverse situation57 , validating the proposed model for CN- transfer occurring by the action of HypE in [NiFe]-hydrogenase active site biosynthesis and drawing an interesting parallel between geochemical and biochemical ligand addition. A further possible parallel between geochemical and biochemical events exists in the possibility of small molecule formation by the condensation of these modifiers. In an extension of chemistry in which CO and CN- become bound to iron, the reverse reaction could take part in the generation of the amino acid glycine. In the [FeFe]-hydrogenase maturation hypothesis outlined above69 , glycine is proposed to be the CO and CN- ligand precursor; a reverse process in which CO and CN- bound to iron condense at the mineral–solution interface with water would result in its formation (Fig. 5b), illustrating the possibility that both the formation as well as the degradation of prebiotic molecules at mineral surfaces could have had important consequences at the origin of life. CO and CN- addition to iron in possible prebiotic conditions may have thus acted as a source for the accumulation of biologically accessible molecules such as amino acids, and as described further below, as a driving force for the formation of diverse catalytic functionality acting as metal surface modifying agents via ligand addition. The challenge of the formation of a diverse repertoire of catalysts has been addressed in part by a proposal based off of ligand modification. Here, the chemical reactions that lead to metal ligand modification are viewed as acting in a type of chemical evolution where catalytically derived products may themselves add to metal centers in chemistry analogous to that described above, modifying their reactivity perhaps for their betterment and thus 4282 | Dalton Trans., 2009, 4274–4285 act to diversify and enhance catalytic attributes.89 This process, in which the addition of a specific ligand leads to a quickening of a particular reaction is termed “ligand accelerated catalysis” and is applicable to both heterogeneous and homogeneous catalysts, being especially relevant to transition metal based catalysts that may exist in environments in which dynamic ligand exchange events are possible.90 The series of events in which ligand modification may serve as a route to positive feed back, termed ligand-accelerated autocatalysis, has been proposed to operate as a most basic type of reproductive mechanism89 and may function in a manner enabling catalytic mineral core derived products to modify clusters thereby producing generations and families of catalysts (Fig. 5c). Indeed, this type of phenomenon may occur in a system wide sense, with neighbouring mineral sites of differing catalytic proclivity contributing to the formation of proficient neighbouring catalysts, thus forming a network basis for catalytic fecundity. Also relevant to the discussion of what events could comprise catalyst formation is the notion of “defect” sites or areas of discontinuity of bulk structure on mineral surfaces. At these locations, which represent approximately 1% of total surface area,91,92 defects in lattice structure result in unique electronic as well as spatial localities, where molecules may transiently bind in orientations and environments amenable to reaction progress. Heterogeneous catalysis by mineral surfaces has indeed been observed to occur at these sites93,94 and highlights the observation of biological clusters existing as structurally, stoichiometrically, and molecularly tuned derivatives of minerals. In addition to the possibility of pre-existing functionality of minerals and ligand modification reactions as having played a role in the synthesis of relevant biological constituents, the possibility of individual iron sulfur clusters in solution exists. Iron–sulfur clusters of the nature [Fe4 S4 (SR)]2- have been shown to assemble in aqueous solvent from iron, sulfide, and thiols (RSH).95 Together with ligand modification schemes and reported experiments showing the generation of iron carbonyls and reactive precursors obtained through hydrothermal reaction conditions80 , these experiments show that a suite of chemistries and possible This journal is © The Royal Society of Chemistry 2009 catalyst generating events closely related to those observed in biology are possible in putative prebiotic conditions and indicate that relevant transition metal based chemistry can occur outside of proteins. Given this, it is interesting to speculate on the catalytic properties of these modified metal species and the possibility that cluster assembly events similar to those that occur biologically to create the active site architecture of the hydrogenases and other metalloproteins may at least be partly mimicked by naturally occurring geochemical processes. Based on the above discussion of synthetic, biosynthetic, and prebiotic reactions, a putative route to biological catalyst creation and the steps associated with this are figuratively portrayed in Fig. 6 starting from an assortment of FeS mineral surfaces. Broadly divided into stages these transitions include the concepts of (i) a mineral basis for early catalysis, (ii) the formation of individual catalytic entities; ligand modification and cluster formation, and (iii) the encapsulation of the nascent metal sulfide clusters by peptides. Specifically, the proposed process of catalyst evolution is portrayed to include the inherent activities of mineral surfaces in which the surface itself or its transformation (oxidation in the case of pyrite formation) results in relevant reactivity (e.g. ref. 78,96– 98). Also at this stage is represented the aforementioned concept of “ready made”84 clusters and defect sites which may catalyze reactions contributing to the formation of biologically accessible molecules. With these roles in mind, a next step in catalyst evolution may have been the formation of individual clusters and cluster components such as the di-iron hexacarbonyl species as obtained under hydrothermal conditions upon the reaction with CO and alkyl thiols80,99 , the formation of iron sulfur cubanes, and the formation of Fe–CN bonds as observed in spark discharge experiments. The integration of these concepts and chemistries may lead to the generation of proto-metallo-co-factors which bear similarity to those observed in biology today. These intermediary clusters may, in a similar way as described by Milner-White and Russell, be bound by peptide “nests” comprised of simple amino acids.100,101 Finally, the incorporation of these clusters into more complex peptide chains leads to protein bound catalysts capable of supporting catalytic activity from a robust mobile peptide platform and the formation of an early proto-metabolism replete with catalysts of sufficient diversity by which to harness chemical energy. Prospectus The structural and spectroscopic characterization of the hydrogenase enzymes created new lines of research that, in an interesting complementarity, are applicable both to future energy solutions and to understanding the past evolution of hydrogen redox catalysts and their role in the formation of life. With the rapid pace of research in this area, the field is certain to yield additional new insights in the coming years. For example, much is yet to be revealed regarding biosynthesis of the metal centers of the hydrogenases, particularly the [FeFe] and [Fe] classes. In addition, while synthetic studies have resulted in the development of creative and often elegant chemistries, an effective first row transition metal based catalyst for hydrogen production based on the structure of hydrogenase active sites has not emerged, perhaps hinting that the proteinaceous environments surrounding the active sites of the hydrogenases are critical for fine-tuning This journal is © The Royal Society of Chemistry 2009 Fig. 6 Possible set of major transition stages in the emergence of prebiotically relevant catalysts from metal sulfide minerals. Evolutionary routes to protein bound transition metal sulfide catalysts are postulated as having originated from the basis of iron and sulfur bearing minerals. As a first illustrative stage stemming from the inherent properties of metal-sulfides, the lattice structure of pyrite104 is shown as being derived from FeS by oxidation which can serve as a driver for prebiotic catalysis. In addition, the structure of a nickel–iron sulfide is shown which may harbour pre-existing functionality. Also depicted at this stage is the portrayal of a mineral surface defect site where atomic irregularities in the lattice structure may lead to relevant catalytic activity through modified electronic and small molecule binding characteristics. Continuing in catalyst evolution, the next stage involves individual cluster formation and possible ligand modifications in the forms of CO and CN- addition and the creation of individual metal centers resulting in the iron hexacyanide, [4Fe-4S], and di-iron hexacarbonyl species (Fe: dark red, S: orange, O: red, C: dark gray, R group: magenta, N: blue) resulting in an intermediary step prior to the protein encapsulation of metal co-factors. In the final stage (top), the combination of these chemistries leads to similar metal clusters to those seen in biology today, being bound by relatively simple peptides which serve to contribute the unique solvent micro-environment observed in contemporary metalloenzymes and exist as ligand modifying agents themselves. Shown as examples are the metal cluster active sites of acetyl-CoA synthase105 , the [FeFe]-hydrogenase, and carbon monoxide dehydrogenase.106 Together, these representations illustrate only a few of the myriad possibilities and routes available in the formation of transition metal based catalytic species. the catalytic potential of the metal centers, making studies in which synthetic metal co-factors are linked to designed peptides particularly attractive.102 Given their high catalytic efficiencies, hydrogenase enzymes themselves remain interesting prospects for biotechnology for non-noble metal based hydrogen oxidation and hydrogen production catalysis in a hydrogen fuel economy and a thorough understanding of the structural determinants of catalysis and the biosynthesis of these enzymes is required for the full potential to be realized. Finally, the ideas of preformed geologically derived catalytic motifs, ligand accelerated autocatalysis, Dalton Trans., 2009, 4274–4285 | 4283 and the chemical ability of catalysts derived as such, may together prove to yield deep insight into the origins of modern biological complexity. The brief account of possibilities concerning prebiotic catalysis described herein indicates that much is possible in relevant primordial conditions with concern to cluster assembly and ligand modification. The observations presented demonstrate a diverse array of modifications that minerals and individual metal ions may undergo and indicate that studies into transition metal minerals and the possible transformations possible from these may be a highly insightful area of research with concern to prebiotic catalysis. Continued work directed at the areas described herein will shed new and fascinating light on hydrogenases; biology’s response to the hydrogen redox couple. 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