Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli.

The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive.

Redox tuning of the H-cluster by second coordination sphere amino acids in the sensory [FeFe] hydrogenase from Thermotoga maritima.

[FeFe] hydrogenases are exceptionally active catalysts for the interconversion of molecular hydrogen with protons and electrons. Their active site, the H-cluster, is composed of a [4Fe-4S] cluster covalently linked to a unique [2Fe] subcluster. These enzymes have been extensively studied to understand how the protein environment tunes the properties of the Fe ions for efficient catalysis. The sensory [FeFe] hydrogenase (HydS) from Thermotoga maritima has low activity and displays a very positive redox potential for the [2Fe] subcluster compared to that of the highly active prototypical enzymes. Using site directed mutagenesis, we investigate how second coordination sphere interactions of the protein environment with the H-cluster in HydS influence the catalytic, spectroscopic and redox properties of the H-cluster. In particular, mutation of the non-conserved serine 267, situated between the [4Fe-4S] and [2Fe] subclusters, to methionine (conserved in prototypical catalytic enzymes) gave a dramatic decrease in activity. Infra-red (IR) spectroelectrochemistry revealed a 50 mV lower redox potential for the [4Fe-4S] subcluster in the S267M variant. We speculate that this serine forms a hydrogen bond to the [4Fe-4S] subcluster, increasing its redox potential. These results demonstrate the importance of the secondary coordination sphere in tuning the catalytic properties of the H-cluster in [FeFe] hydrogenases and reveal a particularly important role for amino acids interacting with the [4Fe-4S] subcluster.

Binding of exogenous cyanide reveals new active-site states in [FeFe] hydrogenases.

[FeFe] hydrogenases are highly efficient metalloenyzmes for hydrogen conversion. Their active site cofactor (the H-cluster) is composed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) linked to a unique organometallic di-iron subcluster ([2Fe]H). In [2Fe]H the two Fe ions are coordinated by a bridging 2-azapropane-1,3-dithiolate (ADT) ligand, three CO and two CN- ligands, leaving an open coordination site on one Fe where substrates (H2 and H+) as well as inhibitors (e.g. O2, CO, H2S) may bind. Here, we investigate two new active site states that accumulate in [FeFe] hydrogenase variants where the cysteine (Cys) in the proton transfer pathway is mutated to alanine (Ala). Our experimental data, including atomic resolution crystal structures and supported by calculations, suggest that in these two states a third CN- ligand is bound to the apical position of [2Fe]H. These states can be generated both by "cannibalization" of CN- from damaged [2Fe]H subclusters as well as by addition of exogenous CN-. This is the first detailed spectroscopic and computational characterisation of the interaction of exogenous CN- with [FeFe] hydrogenases. Similar CN--bound states can also be generated in wild-type hydrogenases, but do not form as readily as with the Cys to Ala variants. These results highlight how the interaction between the first amino acid in the proton transfer pathway and the active site tunes ligand binding to the open coordination site and affects the electronic structure of the H-cluster.

Investigating the role of the strong field ligands in [FeFe] hydrogenase: spectroscopic and functional characterization of a semi-synthetic mono-cyanide active site.

Artificial maturation of hydrogenases provides a path towards generating new semi-synthetic enzymes with novel catalytic properties. Here enzymes featuring a synthetic asymmetric mono-cyanide cofactor have been prepared using two different hydrogenase scaffolds. Their structure and reactivity was investigated in order to elucidate the design rationale behind the native di-cyanide cofactor, and by extension the second coordination sphere of the active-site pocket. Surprisingly, the choice of host enzyme was found to have a dramatic impact on reactivity. Moreover, the study shows that synthetic manipulations of the active-site can significantly increase inhibitor tolerance, as compared to native [FeFe] hydrogenase, while retaining the enzyme's native capacity for reversible catalysis.

Bioelectrocatalytic CO2 Reduction by Redox Polymer-Wired Carbon Monoxide Dehydrogenase Gas Diffusion Electrodes.

The development of electrodes for efficient CO2 reduction while forming valuable compounds is critical. The use of enzymes as catalysts provides the advantage of high catalytic activity in combination with highly selective transformations. We describe the electrical wiring of a carbon monoxide dehydrogenase II from Carboxydothermus hydrogenoformans (ChCODH II) using a cobaltocene-based low-potential redox polymer for the selective reduction of CO2 to CO over gas diffusion electrodes. High catalytic current densities of up to -5.5 mA cm-2 are achieved, exceeding the performance of previously reported bioelectrodes for CO2 reduction based on either carbon monoxide dehydrogenases or formate dehydrogenases. The proposed bioelectrode reveals considerable stability with a half-life of more than 20 h of continuous operation. Product quantification using gas chromatography confirmed the selective transformation of CO2 into CO without any parasitic co-reactions at the applied potentials.

Stability of the H-cluster under whole-cell conditions-formation of an Htrans-like state and its reactivity towards oxygen.

Hydrogenases are metalloenzymes that catalyze the reversible oxidation of molecular hydrogen into protons and electrons. For this purpose, [FeFe]-hydrogenases utilize a hexanuclear iron cofactor, the H-cluster. This biologically unique cofactor provides the enzyme with outstanding catalytic activities, but it is also highly oxygen sensitive. Under in vitro conditions, oxygen stable forms of the H-cluster denoted Htrans and Hinact can be generated via treatment with sulfide under oxidizing conditions. Herein, we show that an Htrans-like species forms spontaneously under intracellular conditions on a time scale of hours, concurrent with the cells ceasing H2 production. Addition of cysteine or sulfide during the maturation promotes the formation of this H-cluster state. Moreover, it is found that formation of the observed Htrans-like species is influenced by both steric factors and proton transfer, underscoring the importance of outer coordination sphere effects on H-cluster reactivity.

The Nonphysiological Reductant Sodium Dithionite and [FeFe] Hydrogenase: Influence on the Enzyme Mechanism.

[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H2). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) covalently attached to a unique [2Fe] subcluster ([2Fe]H), where both sites are redox active. Heterolytic splitting and formation of H2 takes place at [2Fe]H, while [4Fe-4S]H stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state Hox, named HoxH, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. HoxH was previously suggested to have a protonated [4Fe-4S]H. Here, we show that HoxH forms by simple addition of sodium sulfite (Na2SO3, the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO2) is the species involved. Spectroscopy supports binding at or near [4Fe-4S]H, causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that HoxH and its one-electron reduced counterpart Hred'H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the "dithionite (DT) inhibited" states Hox-DTi and Hred-DTi. The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.

Caught in the Hinact : Crystal Structure and Spectroscopy Reveal a Sulfur Bound to the Active Site of an O2 -stable State of [FeFe] Hydrogenase.

[FeFe] hydrogenases are the most active H2 converting catalysts in nature, but their extreme oxygen sensitivity limits their use in technological applications. The [FeFe] hydrogenases from sulfate reducing bacteria can be purified in an O2 -stable state called Hinact . To date, the structure and mechanism of formation of Hinact remain unknown. Our 1.65 Šcrystal structure of this state reveals a sulfur ligand bound to the open coordination site. Furthermore, in-depth spectroscopic characterization by X-ray absorption spectroscopy (XAS), nuclear resonance vibrational spectroscopy (NRVS), resonance Raman (RR) spectroscopy and infrared (IR) spectroscopy, together with hybrid quantum mechanical and molecular mechanical (QM/MM) calculations, provide detailed chemical insight into the Hinact state and its mechanism of formation. This may facilitate the design of O2 -stable hydrogenases and molecular catalysts.

Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase.

The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H+) by dihydronicotinamide adenine dinucleotide (NADH) (∆G0 ≈ 18 kJ mol-1) to the exergonic reduction of H+ by reduced ferredoxin (Fdred) (∆G0 ≈ - 16 kJ mol-1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization of TmHydABC recombinantly produced in Escherichia coli and artificially maturated with a synthetic diiron cofactor. We found that TmHydABC catalyzed the hydrogen (H2)-dependent reduction of nicotinamide adenine dinucleotide (NAD+) in the presence of oxidized ferredoxin (Fdox) at a rate of ≈17 µmol NADH min-1 mg-1. Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H2 conversion, the H-cluster, in TmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases.

His-Ligation to the [4Fe-4S] Subcluster Tunes the Catalytic Bias of [FeFe] Hydrogenase.

[FeFe] hydrogenases interconvert H2 into protons and electrons reversibly and efficiently. The active site H-cluster is composed of two sites: a unique [2Fe] subcluster ([2Fe]H) covalently linked via cysteine to a canonical [4Fe-4S] cluster ([4Fe-4S]H). Both sites are redox active and electron transfer is proton-coupled, such that the potential of the H-cluster lies very close to the H2 thermodynamic potential, which confers the enzyme with the ability to operate quickly in both directions without energy losses. Here, one of the cysteines coordinating [4Fe-4S]H (Cys362) in the [FeFe] hydrogenase from the green algae Chlamydomonas reinhardtii ( CrHydA1) was exchanged with histidine and the resulting C362H variant was shown to contain a [4Fe-4S] cluster with a more positive redox potential than the wild-type. The change in the [4Fe-4S] cluster potential resulted in a shift of the catalytic bias, diminishing the H2 production activity but giving significantly higher H2 oxidation activity, albeit with a 200 mV overpotential requirement. These results highlight the importance of the [4Fe-4S] cluster as an electron injection site, modulating the redox potential and the catalytic properties of the H-cluster.

Sulfide Protects [FeFe] Hydrogenases From O2.

[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation with high rates and efficiency under physiological conditions, but are highly oxygen sensitive. The [FeFe] hydrogenase from Desulfovibrio desulfuricans ( DdHydAB) can be purified under air in an oxygen stable inactive state Hoxair. The formation of the Hoxair state in vitro allows the handling of hydrogenases in air, making their implementation in biotechnological applications more feasible. Here, we report a simple and robust protocol for the formation of the Hoxair state in DdHydAB and the [FeFe] hydrogenase from Chlamydomonas reinhardtii, which is based on high potential inactivation in the presence of sulfide.

Dual properties of a hydrogen oxidation Ni-catalyst entrapped within a polymer promote self-defense against oxygen.

The Ni(P2N2)2 catalysts are among the most efficient non-noble-metal based molecular catalysts for H2 cycling. However, these catalysts are O2 sensitive and lack long term stability under operating conditions. Here, we show that in a redox silent polymer matrix the catalyst is dispersed into two functionally different reaction layers. Close to the electrode surface is the "active" layer where the catalyst oxidizes H2 and exchanges electrons with the electrode generating a current. At the outer film boundary, insulation of the catalyst from the electrode forms a "protection" layer in which H2 is used by the catalyst to convert O2 to H2O, thereby providing the "active" layer with a barrier against O2. This simple but efficient polymer-based electrode design solves one of the biggest limitations of these otherwise very efficient catalysts enhancing its stability for catalytic H2 oxidation as well as O2 tolerance.

Intercluster Redox Coupling Influences Protonation at the H-cluster in [FeFe] Hydrogenases.

[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation displaying high rates at low overpotential. Their active site is a complex cofactor consisting of a unique [2Fe] subcluster ([2Fe]H) covalently bound to a canonical [4Fe-4S] cluster ([4Fe-4S]H). The [FeFe] hydrogenase from Desulfovibrio desulfuricans is exceptionally active and bidirectional. This enzyme features two accessory [4Fe-4S]F clusters for exchanging electrons with the protein surface. A thorough understanding of the mechanism of this efficient enzyme will facilitate the development of synthetic molecular catalysts for hydrogen conversion. Here, it is demonstrated that the accessory clusters influence the catalytic properties of the enzyme through a strong redox interaction between the proximal [4Fe-4S]F cluster and the [4Fe-4S]H subcluster of the H-cluster. This interaction enhances proton-coupled electronic rearrangement within the H-cluster increasing the apparent pKa of its one electron reduced state. This may help to sustain H2 production at high pH values. These results may apply to all [FeFe] hydrogenases containing accessory clusters.

Spectroscopic Evidence of Reversible Disassembly of the [FeFe] Hydrogenase Active Site.

[FeFe] hydrogenases are extremely active and efficient H2-converting biocatalysts. Their active site comprises a unique [2Fe] subcluster bonded to a canonical [4Fe-4S] cluster. The [2Fe] subsite can be introduced into hydrogenases lacking an assembled H-cluster through incubation with a synthesized [2Fe]H precursor, which initially produces the CO-inhibited state of the enzyme. We present FTIR spectroelectrochemical studies on the CO-inhibited state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans, DdHydAB. At very negative potentials, disassembly of the H-cluster and dissociation of the [2Fe] subcluster is observed. Subsequently raising the potential allows cofactor rebinding and H-cluster reassembly. This demonstrates how the stability of the [2Fe]-[4Fe-4S] intercluster bond depends on the applied potential and the presence of an inhibiting CO ligand on the [2Fe] subcluster. These results provide insight into the mechanisms of CO inhibition and H-cluster assembly in [FeFe] hydrogenases. A fundamental understanding of these properties will provide clues for designing better H2-converting catalysts.

The structurally unique photosynthetic Chlorella variabilis NC64A hydrogenase does not interact with plant-type ferredoxins.

Hydrogenases from green algae are linked to the photosynthetic electron transfer chain via the plant-type ferredoxin PetF. In this work the [FeFe]-hydrogenase from the Trebouxiophycean alga Chlorella variabilis NC64A (CvHydA1), which in contrast to other green algal hydrogenases contains additional FeS-cluster binding domains, was purified and specific enzyme activities for both hydrogen (H2) production and H2 oxidation were determined. Interestingly, although C. variabilis NC64A, like many Chlorophycean algal strains, exhibited light-dependent H2 production activity upon sulfur deprivation, CvHydA1 did not interact in vitro with several plant-type [2Fe-2S]-ferredoxins, but only with a bacterial2[4Fe4S]-ferredoxin. In an electrochemical characterization, the enzyme exhibited features typical of bacterial [FeFe]-hydrogenases (e.g. minor anaerobic oxidative inactivation), as well as of algal enzymes (very high oxygen sensitivity).

Influence of the [4Fe-4S] cluster coordinating cysteines on active site maturation and catalytic properties of C. reinhardtii [FeFe]-hydrogenase.

[FeFe]-Hydrogenases catalyze the evolution and oxidation of hydrogen using a characteristic cofactor, termed the H-cluster. This comprises an all cysteine coordinated [4Fe-4S] cluster and a unique [2Fe] moiety, coupled together via a single cysteine. The coordination of the [4Fe-4S] cluster in HydA1 from Chlamydomonas reinhardtii was altered by single exchange of each cysteine (C115, C170, C362, and C366) with alanine, aspartate, or serine using site-directed mutagenesis. In contrast to cysteine 115, the other three cysteines were found to be dispensable for stable [4Fe-4S] cluster incorporation based on iron determination, UV/vis spectroscopy and electron paramagnetic resonance. However, the presence of a preformed [4Fe-4S] cluster alone does not guarantee stable incorporation of the [2Fe] cluster. Only variants C170D, C170S, C362D, and C362S showed characteristic signals for an inserted [2Fe] cluster in Fourier-transform infrared spectroscopy. Hydrogen evolution and oxidation were observed for these variants in solution based assays and protein-film electrochemistry. Catalytic activity was lowered for all variants and the ability to operate in either direction was also influenced.

Electrochemical Investigations on the Inactivation of the [FeFe] Hydrogenase from Desulfovibrio desulfuricans by O2 or Light under Hydrogen-Producing Conditions.

The applicability of the extremely active [FeFe] hydrogenase from Desulfovibrio desulfuricans in H2 -producing devices is studied. Despite being the most active enzyme for H2 catalysis, its high sensitivity towards O2 has prevented its use in electrolytic water splitting cells. Using electrochemical methods, the catalytic activity of the enzyme at H2 -producing potentials and its inactivation upon exposure to limited amounts of O2 or under illumination is analysed. This enzyme is shown to maintain H2 production activity for extended periods of time at low potentials. At such potentials, the enzyme can deliver the required electrons to fully reduce O2 to H2 O, minimising the damage of the enzyme. Additionally, the robustness of this enzyme under illumination at negative applied potentials is demonstrated. These results show that the [FeFe] hydrogenase from D. desulfuricans is an excellent candidate to be used in devices to store solar energy in hydrogen.

Direct comparison of the performance of a bio-inspired synthetic nickel catalyst and a [NiFe]-hydrogenase, both covalently attached to electrodes.

The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we have come in mimicking the enzymatic performance. The catalytic properties of the [Ni(P(Cy) 2 N(Gly) 2 )2 ](2+) complex with the [NiFe]-hydrogenase from Desulfovibrio vulgaris immobilized on a functionalized electrode were compared under identical conditions. At pH 7, the enzyme shows higher activity and lower overpotential with better stability, while at low pH, the molecular catalyst outperforms the enzyme in all respects. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bio-inspired complexes in fuel cells.