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ConspectusThe well-known dinuclear [FeFe] and [NiFe] hydrogenase enzymes are redox-based proton reduction and H2 oxidation catalysts. In comparison, the structural and functional aspects of the mon...

Chemical Science, 12 (32), ISSN:2041-6520, ISSN:2041-6539

A combined experimental and computational investigation of the CO dissociation properties of three [Fe]-hydrogenase models, [(2-CH2CO-6-HOC5H3N)Fe(CO)3I] (1), [(2-CH2CO-6-MeOC5H3N)Fe(CO)3I] (2), and [(2-CH2CO-6-t-BuOC5H3N)Fe(CO)3I] (3), shows equilibria of tricarbonyl and dicarbonyl complexes in solution. In CH3CN, 1 transforms to the solvated product [(2-CH2CO-6-HOC5H3N)Fe(CO)2(CH3CN)I] (7). The reactivity of 2 with PPh3 was also explored, giving [(2-CH2CO-6-MeOC5H3N)Fe(CO)2(PPh3)I] (8) via one CO substitution by PPh3.

The ruthenacyclic carbamoyl complexes [RuBr(2-NHC(O)C5H3N-4-R)(CO)2(MeCN)] in which the 4-position of the pyridinyl ring is derivatized, have been synthesized. These are ruthenium-based structural mimics of the natural [Fe]-hydrogenase metal cofactor. The derivative with R = H reacts with phosphine to form a disubstituted product [RuBr(2-NHC(O)C5H4N)(CO)(PPh2SPh)2].

Hydrogenase-viologen-porphyrin triad was immobilized on ITO electrode and carried out photoinduced hydrogen evolution. This system can reductively generate hydrogen by electron donation from the photoexcited porphyrin to hydrogenase via viologen. Hydrogen production by water splitting (electrochemical, photochemical, or thermal) offers an obvious way to capture solar energy. Catalysts are a crucial issue for Hydrogen production, and there is intense interest in finding alternatives to noble metals that are capable of converting water into hydrogen close to the thermodynamic potential. As a biological alternative, enzymes known as hydrogenases catalyze the reduction of protons into hydrogen at active sites composed of iron- or nickel/iron complexes: importantly, the electrochemical reaction is reversible with a small overpotential. Determination of the structures of hydrogenases from several organisms paired with detailed spectroscopic and electrochemical investigations have considerably improved our understanding of these enzymes in recent years. Hydrogenases also show high H2 production yields in photocatalytic schemes within sacrificial electron donors in pH neutral aqueous solution. In these systems with hydrogenase, a photoexcited photosensitizer provides electrons to the active site where proton reduction occurs via an internal electron transfer iron sulfur clusters. Examples are the immobilization of a hydrogenase on Ru-sensitised TiO2, on Cd-based, quantum dotsas well as homogeneous systems using the hydrogenases with a covalently linked photosystem I or in combination with an organic dye,and multi-component systems with a dye and a soluble redox mediator. In this study, we report on ITO electrode system with hydrogenase, where Ni-Fe hydrogenase immobilized on ITO surface using a linker contains both a viologen and a porphyrin. [NiFe]-Hydrogenase from Desulfovibrio vulgaris (Miyazaki) is approximately 90 kDa in size, with large and small subunits of 60 kDa and 30 kDa, respectively. The Ni-Fe active site is located to the surface of the enzyme. Three Fe-S clusters from the putative electron transfer pathway between the active site. The proximal [4Fe4S] cluster lies closest to the active site while the distal [4Fe4S] cluster is found near the surface of the enzyme. A [3Fe4S] cluster is located in between the two [4Fe4S] clusters and is known as the medial cluster. The distal [4Fe4S] cluster works as the first electron acceptor of the [NiFe]-hydrogenase in the case of hydrogen evolution. Figure 1 shows the ITO electrode system prepared in this study. Hydrogenase was immobilized via the 4,4’-(3-aminopropyl)-bipyridinium (DAPV) by a glutamate-rich patch of hydrogenase where the distal Fe-S cluster is located. The photoexcited state of Tetrakis(4-carboxyphenyl)porphyrin (TCPP) donates electrons to [NiFe]-hydrogenase via the 4,4’-(3-aminopropyl)-bipyridinium (DAPV) bridging between the two. DAPV was linked to acidic amino acid near the distal [4Fe4S] cluster, electron entrance, of [NiFe]-Hydrogenase in order to efficiently donate electrons to the [NiFe]-Hydrogenase. Photoinduced hydrogen evolution were carried out under applied -0.3V vs.SHE. During light irradiation, cathodic continuous current was observed due to the hydrogen evolution. The photoexcited state of TCPP donate electrons to hydrogenase via the viologen, and ITO electrode play an electron donor to HOMO of the TCPP. Therefore, photoinduced hydrogen evolution occur in TCPP-DAPV-hydrogenase triads on ITO electrode. Figure 1

In this work, density functional theory (DFT) calculations were carried out to study the role of solvation effects on the reaction of diiron dithiolate complex with CO to form [Fe]-hydrogenase model complex. In the gas phase, the energy barrier of the first transition state TS 1 species is ca. 6.1 kcal/mol higher than the second transition state TS 2 species. However, when the solvation effects were included, the energy order was reversed, i.e., the energy barrier of TS 1 falls ca. 1.2 kcal/mol lower than TS 2 , indicating that the insertion of the second CO to iron is the rate-determining step in the whole transformation process. The initial insertion of the CO plays an important role in increasing the reaction barrier of the binding of a second CO, which prevented the second step transformation. Thus, the solvation effects play a significant role in determining the reaction mechanism. In addition, the energy of PC species is lower than RC species, demonstrating that this transformation is a significantly exothermic process.

A set of structural mimics of the [Fe]-hydrogenase active site comprising all the group VIII metals, viz., [M(2-NHC(O)C5H4N)(CO)2(2-S-C5H4N)], has been synthesized. They exist as a mixture of isomers in solution, and the relative stability of the isomers depends on the nature of the metal and the substituent at the 6-position of the pyridine ligand. MOE (Min. of Education, S’pore) Accepted version

We have developed two novel synthetic methods, by which two types of mononuclear Fe model complexes for the active site of [Fe]hydrogenase are successfully synthesized. The first type of 2-acylmethyl-6-hydroxymethylpyridine-containing complexes, [2-COCH2-6-HOCH2C5H3N]Fe(CO)2G (1, G = PhCO2; 2, PhCOS; 3, PhCS2; 4, 2-S-6-MeC5H3N), were prepared by a “one-pot” method involving reaction of 2-TsO-6-HOCH2C5H3N (Ts = 4-MeC6H4SO2) with Na2Fe(CO)4 followed by treatment of the resulting Fe(0) intermediate [Na(2-CH2-6-HOCH2C5H3N)Fe(CO)4] (M1) with (PhCO2)2, (PhCOS)2, (PhCS2)2, and (2-S-6-MeC5H3N)2 in 49–72% yields, respectively. The second type of 2-acylmethyl-6-hydroxypyridine-containing complexes, (2-COCH2-6-HOC5H3N)Fe(CO)2(2-SCO-6-RC5H3N) (9a, R = MeO; 9b, R = PhS), could be prepared via a multiple-step synthetic method. This method involves (i) treatment of 2-ClCO-6-RC5H3N (R = MeO, PhS) with NaSH followed by acidification with diluted HCl to give 2-HSCO-6-RC5H3N (5a, R = MeO; 5b, R = PhS); (ii) further treatmen...

On synthesizing the PMB-protected mononuclear iron complexes [2-C(O)CH2-6-PMBOC5H3N]Fe(CO)3I (6) and [2-C(O)CH2-6-PMBOC5H3N]Fe(CO)2(2-SC5H4N) (7), the novel acylmethylpyridinol ligand containing dinuclear iron complex [2-C(O)CH2-6-HOC5H3N]Fe2(CO)4[2′-C(O)CH2-6′-OC5H3N](2-SC5H4N) (9), which is closely related to the active site of [Fe]-hydrogenase, has been prepared unexpectedly via removal of the PMB protecting group from 7 under the action of excess trifluoroacetic acid. The [Fe]-hydrogenase-related complex 9 and its precursor complexes 6 and 7 have been fully characterized by elemental analysis, spectroscopy, and X-ray crystallography.

[Fe] hydrogenase is a hydrogen activating enzyme that features a monoiron active site, which can be well characterized by Mössbauer spectroscopy. Mössbauer spectra have been measured of the CO and CN(-) inhibited species as well as under turnover conditions [Shima, S. et al., J. Am. Chem. Soc., 2005, 127, 10430]. This study presents calculated Mössbauer parameters for various active-site models of [Fe] hydrogenase to provide structural information about the species observed in experiment. Because theoretical Mössbauer spectroscopy requires the parametrization of observables from first-principles calculations (i.e., electric-field gradients and contact densities) to the experimental observables (i.e., quadrupole splittings and isomer shifts), nonrelativistic and relativistic density functional theory methods are parametrized against a reference set of Fe complexes specifically selected for the application to the Fe center in [Fe] hydrogenase. With this methodology, the measured parameters for the CO and CN(-) inhibited complexes can be reproduced. Evidence for the protonation states of the hydroxyl group in close proximity to the active site and for the thiolate ligand, which could participate in proton transfer, is obtained. The unknown resting state measured in the presence of the substrate and under pure H2 atmosphere is identified to be a water-coordinated complex. Consistent with previous assignments based on infrared and X-ray absorption near-edge spectroscopy, all measured Mössbauer data can be reproduced with the active site's iron atom being in oxidation state +2.

[Fe] hydrogenase one of three types of hydrogenases activates molecular hydrogen. Here using DFT computations we examine the electronic elements governing the binding of small ligands to a recently synthesized [Fe] hydrogenase biomimic. Computed reaction free energies indicate that anionic species such as CN and H and p acceptors such as CO bind favourably with the Fe centre. Ligands such as H2O CH3CN and H 2 however do not bind iron. Protonation of an adjacent thiolate ligand on the mimic significantly increases the energies of ligand binding. Additional computational analysis reveals that the degree of electron donation from the ligand to the mimic correlates strongly with overall binding ability. The results give insights into the electronic elements of iron small molecule interaction in these model complexes. © 2013 Wiley VCH Verlag GmbH Co. KGaA Weinheim.

The [Fe]-hydrogenase, also known as “H2-forming methylenetetrahydromethanopterin dehydrogenase” (Hmd) or “iron-sulfur cluster-free hydrogenase”, is the third distinct class of hydrogenase enzyme to be discovered. In the presence of its carbocation substrate, it catalyzes the reversible cleavage of dihydrogen into H+ and H-, placing the later concomitantly onto the substrate molecule. This review describes the emerging chemistry behind this fascinating enzyme.

The synthesis, structure, and reactivity of small-molecule models of [Fe]-hydrogenase are described. These models feature the intriguing acylmethylpyridinyl ligands found exclusively in the enzyme.

The synthesis of a close structural analogue of the active site of [Fe]-hydrogenase is described (see structure; C gray, H dark blue, Fe green, N light blue, O red, S yellow). Nature most probably constructs the five membered ferracyclic ring to poise the 2-hydroxy pyridine substituent in a position to assist the heterolytic cleavage of dihydrogen, and the accessibility of the analogue should now provide opportunities for probing this.

This article sets out to review the chemistry relating to the synthesis of structural and functional analogues of the three classes of hydrogenases. This chemistry has grown explosively over the last 10 or so years since the first X-ray structures of [NiFe]- and [FeFe]-hydrogenase systems were published. There are now some 400 papers covering structural and functional aspects, with the majority of these associated with the di-iron system. As much emphasized in earlier papers

As active site models of [Fe]-hydrogenase, tridentate 2-acylmethyl-6-methoxymethoxy-difunctionalized pyridine-containing complexes η(3)-(2-COCH2-6-MeOCH2OC5H3N)Fe(CO)2(L1) (4, L1 = I; 5, SCN; 6, PhCS2) were prepared via the following multistep reactions: (i) etherification of 2-MeO2C-6-HOC5H3N with ClCH2OMe to give 2-MeO2C-6-MeOCH2OC5H3N (1), (ii) reduction of 1 with NaBH4 to give 2-HOCH2-6-MeOCH2OC5H3N (2), (iii) esterification of 2 with 4-toluenesulfonyl chloride to give 2-TsOCH2-6-MeOCH2OC5H3N (3), (iv) nucleophilic substitution of 3 with Na2Fe(CO)4 followed by treatment of the resulting Fe(0) intermediate Na[(2-CH2-6-MeOCH2OC5H3N)Fe(CO)4] (M1) with I2 to give complex 4, and (v) condensation of 4 with KSCN and PhCS2K to give complexes 5 and 6, respectively. In contrast to the preparation of complexes 4-6, bidentate 2-acylmethyl-6-methoxymethoxy-difunctionalized pyridine-containing model complexes η(2)-(2-COCH2-6-MeOCH2OC5H3N)Fe(CO)2(I)(L2) (7, L2 = PPh3; 8, Cy-C6H11NC) and η(2)-(2-COCH2-6-MeOCH2OC5H3N)Fe(CO)2(L3) (9, L3 = 2-SC5H4N; 10, 8-SC9H6N) were prepared by ligand exchange reactions of 4 with PPh3, Cy-C6H11NC, 2-KSC5H4N, and 8-KSC9H6N, respectively. Particularly interesting is that the tridentate 2,6-bis(acylmethyl)pyridine- and 2-acylmethyl-6-arylthiomethylpyridine-containing model complexes η(3)-[2,6-(COCH2)2C5H3N]Fe(CO)2(L4) (11, L4 = PPh3; 12, CO) and η(3)-2-(COCH2-6-ArSCH2C5H3N)Fe(CO)2(ArS) (13, ArS = PhS; 14, 2-S-5-MeC4H2O) were obtained, unexpectedly, when 2,6-(TsOCH2)2C5H3N reacted with Na2Fe(CO)4 followed by treatment of the resulting mixture with ligands PPh3 and CO or disulfides (PhS)2 and (2-S-5-MeC4H2O)2. Reactions of ligand precursors 3 and 2,6-(TsOCH2)2C5H3N with Na2Fe(CO)4 were monitored by in situ IR spectroscopy, and the possible pathways for producing complexes 4 and 11-14 via intermediates Na[(2-CH2-6-MeOCH2OC5H3N)Fe(CO)4] (M1), Na[(2-CH2-6-TsOCH2C5H3N)Fe(CO)4] (M2), and (2-COCH2-6-CH2C5H3N)Fe(CO)3 (M3) are suggested. New compounds 1-14 were characterized by elemental analysis, spectroscopy, and, for some of them, X-ray crystallography.

A method based on spectroelectrochemistry to monitor intramolecular electron transfer in [Ni–Fe]-hydrogenase was developed. The electron transfer generated a color change in manganese–tetra(4-pyrid...

The iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase contains a prominent iron centre with an acyl-Fe bond and is the only acyl-organometallic iron compound found in nature. Here, we identify the functions of HcgE and HcgF, involved in the biosynthesis of the FeGP cofactor using structure-to-function strategy. Analysis of the HcgE and HcgF crystal structures with and without bound substrates suggest that HcgE catalyses the adenylylation of the carboxy group of guanylylpyridinol (GP) to afford AMP-GP, and subsequently HcgF catalyses the transesterification of AMP-GP to afford a Cys (HcgF)-S-GP thioester. Both enzymatic reactions are confirmed by in vitro assays. The structural data also offer plausible catalytic mechanisms. This strategy of thioester activation corresponds to that used for ubiquitin activation, a key event in the regulation of multiple cellular processes. It further implicates a nucleophilic attack onto the acyl carbon presumably via an electron-rich Fe(0)- or Fe(I)-carbonyl complex in the Fe-acyl formation.

The gene encoding the [Fe]-hydrogenase from the anaerobic bacterium Megasphaera elsdenii has been cloned and sequenced. The gene is monocistronic, in keeping with the protein being a monomer. The translated protein sequence (484 residues, M(r)=53 kDa) comprises a small 2[4Fe-4S] ferredoxin-like domain and a large domain containing the catalytic site. Comparisons with other [Fe]-hydrogenase sequences, including two of which the crystal structures are known, show that the M. elsdenii protein is among the smallest of these enzymes and provide useful indications regarding the basic structural core common to all [Fe]-hydrogenases. It is, nevertheless, to be noted that the genome of Thermotoga maritima encodes a putative [Fe]-hydrogenase that would consist of only 301 residues.

The thioester Ph2PC6H4-2-C(O)SPh reacts with Fe2(CO)9 to give [Ph2PC6H4C(O)]Fe(SPh)(CO)3, a model for the CO-inhibited active site of the enzyme Hmd. This species, which reversibly decarbonylates t...

International audience; Mössbauer spectroscopy is an indispensable spectroscopic technique and analytical tool in iron coordination chemistry. The linear correlation between the electron density at the nucleus ("contact density") and experimental isomer shifts has been used to link calculated contact densities to experimental isomer shifts. Here we have investigated relativistic methods of systematically increasing sophistication, including the eXact 2-Component (X2C) Hamiltonian and a finite-nucleus model, for the calculation of isomer shifts of iron compounds. While being of similar accuracy as the full four-component treatment, X2C calculations are far more efficient. We find that effects of spin-orbit coupling can safely be neglected, leading to further speedup. Linear correlation plots using effective densities rather than contact densities versus experimental isomer shift lead to a correlation constant a = −0.294 a0−3 mm s−1 (PBE functional) which is close to an experimentally derived value. Isomer shifts of similar quality can thus be obtained both with and without fitting, which is not the case if one pursues a priori a non-relativistic model approach. As an application for a biologically relevant system, we have studied three recently proposed [Fe]-hydrogenase intermediates. The structures of these intermediates were extracted from QM/MM calculations using large QM regions surrounded by the full enzyme and a solvation shell of water molecules. We show that a comparison between calculated and experimentally observed isomer shifts can be used to discriminate between different intermediates, whereas calculated atomic charges do not necessarily correlate with Mössbauer isomer shifts. Detailed analysis reveals that the difference in isomer shifts between two intermediates is due to an overlap effect.

International audience; We report the effect of UV-Vis light on the membrane-bound [Ni-Fe] hydrogenase from Aquifex aeolicus under turnover conditions. Using electrochemistry, we show a potential dependent light sensitivity and propose that a light-induced structural change of the [Ni-Fe] active site is related to an enhanced reactivation of the hydrogenase under illumination at high potentials.

[Fe]-hydrogenase is a newly characterized type of hydrogenase. This enzyme heterolytically splits hydrogen in the presence of a natural substrate. The active site of the enzyme contains a mono-iron complex with intriguing ironacyl ligation. Several groups have recently developed ironacyl complexes as synthetic models of [Fe]-hydrogenase. This Focus Review summarizes the studies of this enzyme and its model compounds, with an emphasis on our own research in this area.

The active site of the [Fe]-hydrogenase features an iron(II) centre bearing cis carbonyl groups and a chelating pyridine–acyl ligand. Reproducing these unusual features in synthetic models is an intriguing challenge, which will allow both better understanding of the enzymatic system and more fundamental insight into the coordination modes of iron. By using the carbamoyl group as a surrogate for acyl, we have been able to synthesize a range of ferracyclic complexes. Initial reaction of Fe(CO)4Br2 with 2-aminopyridine yields a complex bearing a labile solvent molecule, which can be replaced by stronger donors bearing phosphorus atoms to produce a number of derivatives. Introduction of a hydroxy group using this method is unsuccessful both with a free OH group and when this is silyl-protected. In contrast, the analogous reactions starting from 2,6-diaminopyridine does allow synthesis of complexes bearing a pendant basic group.

[Fe]-hydrogenase is a newly characterized type of hydrogenase. This enzyme heterolytically splits hydrogen in the presence of a natural substrate. The active site of the enzyme contains a mono-iron complex with intriguing iron-acyl ligation. Several groups have recently developed iron-acyl complexes as synthetic models of [Fe]-hydrogenase. This Focus Review summarizes the studies of this enzyme and its model compounds, with an emphasis on our own research in this area.

The first acylmethyl(hydroxymethyl)pyridine ligand-containing [Fe]hydrogenase model complexes 2-4 have been synthesized starting from the nucleophilic substitution reaction of 2-(4-MeC(6)H(4)SO(3)CH(2))-6-HOCH(2)C(5)H(3)N with Na(2)Fe(CO)(4). While the reaction course for producing complex 3 via the highly unstable intermediate complex 1 is monitored by in situ IR spectroscopy, the isolated model complexes 2-4 are fully characterized.

Hydrogenases catalyse redox reactions with molecular hydrogen, either as substrate or product. The enzymes harness hydrogen as a reductant using metals that are abundant and economical, namely, nickel and iron, and should provide new pointers for the economic use of hydrogen in manmade devices. The most recently discovered and perhaps the most enigmatic of the hydrogenases is the [Fe]-hydrogenase, used by certain microorganisms in the pathway that reduces carbon dioxide to methane. Since its discovery some twenty years ago, [Fe]-hydrogenase has consistently provided structural and mechanistic surprises, often requiring complete re-evaluation of its mechanism of action. This tutorial review combines recent advances in X-ray crystallography and other analytical techniques, as well as in computational studies and in chemical synthesis to provide a platform for understanding this remarkable enzyme type.

[Fe]-hydrogenase is one of the three types of hydrogenases. This enzyme is found in many hydrogenotrophic methanogenic archaea and catalyzes the reversible hydride transfer from H2 to methenyl-H4MPT+ in methanogenesis from H2 and CO2. The enzyme harbors a unique iron-guanylyl pyridinol (FeGP) cofactor as a prosthetic group. Here, we describe the purification of [Fe]-hydrogenase from Methanothermobacter marburgensis, the isolation of the FeGP cofactor from the native holoenzyme, and the reconstitution of [Fe]-hydrogenase from the isolated FeGP cofactor and the heterologously produced apoenzyme.

Infrared data for mono-iron complexes possessing two cis-CO together with Mossbauer data for the enzyme and a model complex support the assignment that the iron centre of the cluster-free hydrogenase Hmd is low-spin Fe(II).

Recently, a novel Fe-hydrogenase from a high rate of hydrogen producing Enterobacter cloacae strain IIT-BT08 was identified and partially characterized. This 147 residue protein was found to be much smaller than previously known Fe-hydrogenases, yet retaining a high catalytic activity. We predicted the structure of this protein and found it to be structurally similar to one of the two sub-domains containing the catalytic H-cluster so far jointly present in all other Fe-hydrogenases. This novel architecture allows a tentative explanation of protein function with the high rate of catalytic activity being due to a missing regulatory sub-domain, presumably allowing higher enzymatic activity at the cost of greater exposure to oxygen inactivation. This new insight may improve our understanding of the molecular and functional organization of other, more complex Fe-hydrogenases.

Optimized structures for the redox species of the diiron active site in [Fe]-hydrogenase as observed by FTIR and for species in the catalytic cycle for the reversible H(2) oxidation have been determined by density-functional calculations on the active site model, [(L)(CO)(CN)Fe(mu-PDT)(mu-CO)Fe(CO)(CN)(L')](q)(L = H(2)O, CO, H(2), H(-); PDT = SCH(2)CH(2)CH(2)S, L' = CH(3)S(-), CH(3)SH; q = 0, 1-, 2-, 3-). Analytical DFT frequencies on model complexes (mu-PDT)Fe(2)(CO)(6) and [(mu-PDT)Fe(2)(CO)(4)(CN)(2)](2)(-) are used to calibrate the calculated CN(-) and CO frequencies against the measured FTIR bands in these model compounds. By comparing the predicted CN(-) and CO frequencies from DFT frequency calculations on the active site model with the observed bands of D. vulgaris [Fe]-hydrogenase under various conditions, the oxidation states and structures for the diiron active site are proposed. The fully oxidized, EPR-silent form is an Fe(II)-Fe(II) species. Coordination of H(2)O to the empty site in the enzyme's diiron active center results in an oxidized inactive form (H(2)O)Fe(II)-Fe(II). The calculations show that reduction of this inactive form releases the H(2)O to provide an open coordination site for H(2). The partially oxidized active state, which has an S = (1)/(2) EPR signal, is an Fe(I)-Fe(II) species. Fe(I)-Fe(I) species with and without bridging CO account for the fully reduced, EPR-silent state. For this fully reduced state, the species without the bridging CO is slightly more stable than the structure with the bridging CO. The correlation coefficient between the predicted CN(-) and CO frequencies for the proposed model species and the measured CN(-) and CO frequencies in the enzyme is 0.964. The proposed species are also consistent with the EPR, ENDOR, and Mössbauer spectroscopies for the enzyme states. Our results preclude the presence of Fe(III)-Fe(II) or Fe(III)-Fe(III) states among those observed by FTIR. A proposed reaction mechanism (catalytic cycle) based on the DFT calculations shows that heterolytic cleavage of H(2) can occur from (eta(2)-H(2))Fe(II)-Fe(II) via a proton transfer to "spectator" ligands. Proton transfer to a CN(-) ligand is thermodynamically favored but kinetically unfavorable over proton transfer to the bridging S of the PDT. Proton migration from a metal hydride to a base (S, CN, or basic protein site) results in a two-electron reduction at the metals and explains in part the active site's dimetal requirement and ligand framework which supports low-oxidation-state metals. The calculations also suggest that species with a protonated Fe-Fe bond could be involved if the protein could accommodate such species.

Various states or forms of the active site in Ni-Fe hydrogenase, both catalytically active and inactive forms, have been identified and investigated experimentally. Until recently, the geometric structure of each form remained an open question. Several recent theoretical studies with density functional theory have attempted to redress this deficiency. In this commentary, the similarities and differences among the structures proposed by these studies will be addressed.

A nonaheme cytochrome c was purified to homogeneity from the soluble and the membrane fractions of the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex. The gene encoding for the protein was cloned and sequenced. The primary structure of the multiheme protein was highly homologous to that of the nonaheme cytochrome c from D. desulfuricans ATCC 27774 and to that of the 16-heme HmcA protein from Desulfovibrio vulgaris Hildenborough. The analysis of the sequence downstream of the gene encoding for the nonaheme cytochrome c from D. desulfuricans Essex revealed an open reading frame encoding for an HmcB homologue. This operon structure indicated the presence of an Hmc complex in D. desulfuricans Essex, with the nonaheme cytochrome c replacing the 16-heme HmcA protein found in D. vulgaris. The molecular and spectroscopic parameters of nonaheme cytochrome c from D. desulfuricans Essex in the oxidized and reduced states were analyzed. Upon reduction, the pI of the protein changed significantly from 8.25 to 5.0 when going from the Fe(III) to the Fe(II) state. Such redox-induced changes in pI have not been reported for cytochromes thus far; most likely they are the result of a conformational rearrangement of the protein structure, which was confirmed by CD spectroscopy. The reactivity of the nonaheme cytochrome c toward [Ni,Fe] hydrogenase was compared with that of the tetraheme cytochrome c(3); both the cytochrome c(3) and the periplasmic [Ni,Fe] hydrogenase originated from D. desulfuricans Essex. The nonaheme protein displayed an affinity and reactivity toward [Ni,Fe] hydrogenase [K(M) = 20.5 +/- 0.9 microM; v(max) = 660 +/- 20 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)] similar to that of cytochrome c(3) [K(M) = 12.6 +/- 0.7 microM; v(max) = 790 +/- 30 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)]. This shows that nonaheme cytochrome c is a competent physiological electron acceptor for [Ni,Fe] hydrogenase.