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Multicopper oxidases use Cu ions as cofactors to oxidize various substrates. High reduction potential at Type 1 Cu is considered as crucial for effective catalysis. Previous studies have shown that replacing the axial methionine ligand of the Type 1 Cu with leucine or phenylalanine leads to an increased reduction potential, but not always to higher enzyme activity. Here we present a study on six variants of the small laccase Ssl1 from Streptomyces sviceus, where the axial methionine ligand was substituted, and the effect of the axial ligand on reduction potential, activity, spectral properties and structure was investigated. Absorption, electronic circular dichroism and EPR spectra revealed the presence of a stronger coordinating axial ligand like oxygen, which influences the electronic and catalytic properties more than the nature of the amino acid side chain. The crystal structures of the Ssl1 variants were solved, which show that none of the amino acid side chains coordinate to the Cu. Instead, a water molecule is found in the axial coordination site, which support the spectroscopic data. Our findings highlight the importance of combining structural and spectroscopic methods to investigate the effect of amino acid exchange on multicopper oxidases., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

Hydrogen could provide a suitable means for storing energy from intermittent renewable sources for later use on demand. However, many challenges remain regarding the activity, specificity, stability and sustainability of current hydrogen production and consumption methods. The lack of efficient catalysts based on abundant and sustainable elements lies at the heart of this problem. Nature's solution led to the evolution of hydrogenase enzymes capable of reversible hydrogen conversion at high rates using iron- and nickel-based active sites. Through a detailed understanding of these enzymes, we can learn how to mimic them to engineer a new generation of highly active synthetic catalysts. Incredible progress has been made in our understanding of biological hydrogen activation over the last few years. In particular, detailed studies of the [FeFe] hydrogenase class have provided substantial insight into a sophisticated, optimised, molecular catalyst, the active site H-cluster. In this short perspective, we will summarise recent findings and highlight the missing pieces needed to complete the puzzle., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The enzyme FeFe-hydrogenase catalyzes H 2 evolution and oxidation at an active site that consists of a [4Fe-4S] cluster bridged to a [Fe 2 (CO) 3 (CN) 2 (azadithiolate)] subsite. Previous investigations of its mechanism were mostly conducted on a few "prototypical" FeFe-hydrogenases, such as that from Chlamydomonas reinhardtii (Cr HydA1), but atypical hydrogenases have recently been characterized in an effort to explore the diversity of this class of enzymes. We aim at understanding why prototypical hydrogenases are active in either direction of the reaction in response to a small deviation from equilibrium, whereas the homologous enzyme from Thermoanaerobacter mathranii (Tam HydS) shows activity only under conditions of very high driving force, a behavior that was referred to as "irreversible catalysis". We follow up on previous spectroscopic studies and recent developments in the kinetic modeling of bidirectional reactions to investigate and compare the catalytic cycles of Cr HydA1 and Tam HydS under conditions of direct electron transfer with an electrode. We compare the hypothetical catalytic cycles described in the literature, and we show that the observed changes in catalytic activity as a function of potential, pH, and H 2 concentration can be explained with the assumption that the same catalytic mechanism applies. This helps us identify which variations in properties of the catalytic intermediates give rise to the distinct "reversible" or "irreversible" catalytic behaviors.

[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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

[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 (H 2 and H + ) as well as inhibitors ( e.g. O 2 , CO, H 2 S) 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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The development of electrodes for efficient CO 2 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 ( Ch CODH II) using a cobaltocene-based low-potential redox polymer for the selective reduction of CO 2 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 CO 2 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 CO 2 into CO without any parasitic co-reactions at the applied potentials.

Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential pathway to a high-potential acceptor and the other is sent along a low-potential pathway to a low-potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognized, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent 'halves' each made of two strongly interacting HydABC heterotrimers connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron-sulfur cluster domain: a 'closed bridge' and an 'open bridge' conformation, where a Zn 2+ site may act as a 'hinge' allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD + reduction/NADH oxidation site., Competing Interests: CF, NC, PG, WL, HO, JB, JB No competing interests declared, (© 2022, Furlan, Chongdar et al.)

[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe-4S] electron relay covalently attached to the H 2 binding site and a diiron cluster ligated by CO, CN - , and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe-4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe-4S] H but prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.

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 H trans and H inact can be generated via treatment with sulfide under oxidizing conditions. Herein, we show that an H trans -like species forms spontaneously under intracellular conditions on a time scale of hours, concurrent with the cells ceasing H 2 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 H trans -like species is influenced by both steric factors and proton transfer, underscoring the importance of outer coordination sphere effects on H-cluster reactivity., (© 2022. The Author(s).)

Herein, we study the mechanism of iron-catalyzed direct synthesis of unprotected aminoethers from olefins by a hydroxyl amine derived reagent using a wide range of analytical and spectroscopic techniques (Mössbauer, Electron Paramagnetic Resonance, Ultra-Violet Visible Spectroscopy, X-ray Absorption, Nuclear Resonance Vibrational Spectroscopy, and resonance Raman) along with high-level quantum chemical calculations. The hydroxyl amine derived triflic acid salt acts as the "oxidant" as well as "amino" group donor. It activates the high-spin Fe(II) ( S t = 2) catalyst [Fe(acac) 2 (H 2 O) 2 ] ( 1 ) to generate a high-spin ( S t = 5/2) intermediate ( Int I ), which decays to a second intermediate ( Int II ) with S t = 2. The analysis of spectroscopic and computational data leads to the formulation of Int I as [Fe(III)(acac) 2 - N -acyloxy] (an alkyl-peroxo-Fe(III) analogue). Furthermore, Int II is formed by N-O bond homolysis. However, it does not generate a high-valent Fe(IV)(NH) species (a Fe(IV)(O) analogue), but instead a high-spin Fe(III) center which is strongly antiferromagnetically coupled ( J = -524 cm -1 ) to an iminyl radical, [Fe(III)(acac) 2 -NH·], giving S t = 2. Though Fe(NH) complexes as isoelectronic surrogates to Fe(O) functionalities are known, detection of a high-spin Fe(III)- N -acyloxy intermediate ( Int I ), which undergoes N-O bond cleavage to generate the active iron-nitrogen intermediate ( Int II ), is unprecedented. Relative to Fe(IV)(O) centers, Int II features a weak elongated Fe-N bond which, together with the unpaired electron density along the Fe-N bond vector, helps to rationalize its propensity for N -transfer reactions onto styrenyl olefins, resulting in the overall formation of aminoethers. This study thus demonstrates the potential of utilizing the iron-coordinated nitrogen-centered radicals as powerful reactive intermediates in catalysis.

[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H 2 ). 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 H 2 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 H ox , named H ox H, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. H ox H was previously suggested to have a protonated [4Fe-4S] H . Here, we show that H ox H forms by simple addition of sodium sulfite (Na 2 SO 3 , the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO 2 ) 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 H ox H and its one-electron reduced counterpart H red 'H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the "dithionite (DT) inhibited" states H ox -DT i and H red -DT i . The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.

[FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, 57 Fe nuclear resonance vibrational spectroscopy (NRVS), and density functional theory (DFT) calculations to observe and characterize the vibrational modes involving motion of the 2-azapropane-1,3-dithiolate (ADT) ligand bridging the two iron sites in the [2Fe] H subcluster. A - 13 C 2 H 2 - ADT labeling in the synthetic diiron precursor of [2Fe] H produced isotope effects observed throughout the NRVS spectrum. The two precursor isotopologues were then used to reconstitute the H-cluster of [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1), and NRVS was measured on samples poised in the catalytically crucial H hyd state containing a terminal hydride at the distal Fe site. The 13 C 2 H isotope effects were observed also in the H hyd spectrum. DFT simulations of the spectra allowed identification of the 57 Fe normal modes coupled to the ADT ligand motions. Particularly, a variety of normal modes involve shortening of the distance between the distal Fe-H hydride and ADT N-H bridgehead hydrogen, which may be relevant to the formation of a transition state on the way to H 2 formation.

Efficient electrocatalytic energy conversion requires the devices to function reversibly, i.e. deliver a significant current at minimal overpotential. Redox-active films can effectively embed and stabilise molecular electrocatalysts, but mediated electron transfer through the film typically makes the catalytic response irreversible. Here, we describe a redox-active film for bidirectional (oxidation or reduction) and reversible hydrogen conversion, consisting of [FeFe] hydrogenase embedded in a low-potential, 2,2'-viologen modified hydrogel. When this catalytic film served as the anode material in a H 2 /O 2 biofuel cell, an open circuit voltage of 1.16 V was obtained - a benchmark value near the thermodynamic limit. The same film also acted as a highly energy efficient cathode material for H 2 evolution. We explained the catalytic properties using a kinetic model, which shows that reversibility can be achieved despite intermolecular electron transfer being slower than catalysis. This understanding of reversibility simplifies the design principles of highly efficient and stable bioelectrocatalytic films, advancing their implementation in energy conversion., Competing Interests: Competing interests The authors declare no competing interests.

The Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1 365-920 ). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1 371-920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the Fe-S cluster in Asp1 365-920 inside the cell. However, we show that the Fe-S cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S] 2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an Fe-S cluster in vivo that is not involved in its pyrophosphatase activity.

In vivo expression of metalloproteins requires specific metal trafficking and incorporation machinery inside the cell. Synthetic designed metalloproteins are typically purified without the target metal, which is subsequently introduced through in vitro reconstitution. The extra step complicates protein optimization by high-throughput library screening or laboratory evolution. We demonstrate that a designed coiled-coil iron-sulfur protein (CCIS) assembles robustly with [4Fe-4S] clusters in vivo . While in vitro reconstitution produces a mixture of oligomers that depends on solution conditions, in vivo production generates a stable homotrimer coordinating a single, diamagnetic [4Fe-4S] 2+ cluster. The multinuclear cluster of in vivo assembled CCIS is more resistant to degradation by molecular oxygen. Only one of the two metal coordinating half-sites is required in vivo, indicating specificity of molecular recognition in recruitment of the metal cluster. CCIS, unbiased by evolution, is a unique platform to examine iron-sulfur protein biogenesis and develop synthetic multinuclear oxidoreductases.

Oxidoreductase enzymes often perform technologically useful chemical transformations using abundant metal cofactors with high efficiency under ambient conditions. The understanding of the catalytic mechanism of these enzymes is, however, highly dependent on the availability of well-characterized and optimized time-resolved analytical techniques. We have developed an approach for rapidly injecting electrons into a catalytic system using a photoactivated nanomaterial in combination with a range of redox mediators to produce a potential jump in solution, which then initiates turnover via electron transfer (ET) to the catalyst. The ET events at the nanomaterial-mediator-catalyst interfaces are, however, highly sensitive to the experimental conditions such as photon flux, relative concentrations of system components, and pH. Here, we present a systematic optimization of these experimental parameters for a specific catalytic system, namely, [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1). The developed strategies can, however, be applied in the study of a wide variety of oxidoreductase enzymes. Our potential jump system consists of CdSe/CdS core-shell nanorods as a photosensitizer and a series of substituted bipyridinium salts as mediators with redox potentials in the range from -550 to -670 mV (vs SHE). With these components, we screened the effect of pH, mediator concentration, protein concentration, photosensitizer concentration, and photon flux on steady-state photoreduction and hydrogen production as well as ET and potential jump efficiency. By manipulating these experimental conditions, we show the potential of simple modifications to improve the tunability of the potential jump for application to study oxidoreductases.

[FeFe] hydrogenases are the most active H 2 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 O 2 -stable state called H inact . To date, the structure and mechanism of formation of H inact remain unknown. Our 1.65 Å crystal 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 H inact state and its mechanism of formation. This may facilitate the design of O 2 -stable hydrogenases and molecular catalysts., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The incorporation of highly active but also highly sensitive catalysts (e.g. the [FeFe] hydrogenase from Desulfovibrio desulfuricans) in biofuel cells is still one of the major challenges in sustainable energy conversion. We report the fabrication of a dual-gas diffusion electrode H 2 /O 2 biofuel cell equipped with a [FeFe] hydrogenase/redox polymer-based high-current-density H 2 -oxidation bioanode. The bioanodes show benchmark current densities of around 14 mA cm -2 and the corresponding fuel cell tests exhibit a benchmark for a hydrogenase/redox polymer-based biofuel cell with outstanding power densities of 5.4 mW cm -2 at 0.7 V cell voltage. Furthermore, the highly sensitive [FeFe] hydrogenase is protected against oxygen damage by the redox polymer and can function under 5 % O 2 ., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

[FeFe] hydrogenases are highly active hydrogen conversion catalysts but are notoriously sensitive to oxidative damage. Redox hydrogels have been used for protecting hydrogenases from both high potential inactivation and oxygen inactivation under turnover conditions. However, [FeFe] hydrogenase containing redox hydrogels must be fabricated under strict anoxic conditions. Sulfide coordination at the active center of the [FeFe] hydrogenase from Desulfovibrio desulfuricans protects this enzyme from oxygen in an inactive state, which can be reactivated upon reduction. Here, we show that this oxygen-stable inactive form of the hydrogenase can be reactivated in a redox hydrogel enabling practical use of this highly O 2 sensitive enzyme without the need for anoxic conditions.

Nitrogenases are responsible for biological nitrogen fixation, a crucial step in the biogeochemical nitrogen cycle. These enzymes utilize a two-component protein system and a series of iron-sulfur clusters to perform this reaction, culminating at the FeMco active site (M = Mo, V, Fe), which is capable of binding and reducing N 2 to 2NH 3 . In this review, we summarize how different spectroscopic approaches have shed light on various aspects of these enzymes, including their structure, mechanism, alternative reactivity, and maturation. Synthetic model chemistry and theory have also played significant roles in developing our present understanding of these systems and are discussed in the context of their contributions to interpreting the nature of nitrogenases. Despite years of significant progress, there is still much to be learned from these enzymes through spectroscopic means, and we highlight where further spectroscopic investigations are needed.

The active site of [FeFe] hydrogenase features a binuclear iron cofactor Fe 2 ADT(CO) 3 (CN) 2 , where ADT represents the bridging ligand aza-propane-dithiolate. The terminal diatomic ligands all coordinate in a basal configuration, and one CO bridges the two irons leaving an open coordination site at which the hydrogen species and the competitive inhibitor CO bind. Externally supplied CO is expected to coordinate in an apical configuration. However, an alternative configuration has been proposed in which, due to ligand rotation, the CN - bound to the distal Fe becomes apical. Using selective 13 C isotope labeling of the CN - and CO ext ligands in combination with pulsed 13 C electron-nuclear-nuclear triple resonance spectroscopy, spin polarization effects are revealed that, according to density functional theory calculations, are consistent with only the "unrotated" apical CO ext configuration.

The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H + ) by dihydronicotinamide adenine dinucleotide (NADH) (∆G 0  ≈ 18 kJ mol -1 ) to the exergonic reduction of H + by reduced ferredoxin (Fd red ) (∆G 0  ≈ - 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 (H 2 )-dependent reduction of nicotinamide adenine dinucleotide (NAD + ) in the presence of oxidized ferredoxin (Fd ox ) 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 H 2 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.

[FeFe] hydrogenases are extremely active H 2 -converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called H red H + and H sred H + . In one model, the H red H + and H sred H + states contain a semibridging CO, while in the other model, the bridging CO is replaced by a bridging hydride. Using low-temperature IR spectroscopy and nuclear resonance vibrational spectroscopy, together with density functional theory calculations, we show that the bridging CO is retained in the H sred H + and H red H + states in the [FeFe] hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulfuricans , respectively. Furthermore, there is no evidence for a bridging hydride in either state. These results agree with a model of the catalytic cycle in which the H red H + and H sred H + states are integral, catalytically competent components. We conclude that proton-coupled electron transfer between the two subclusters is crucial to catalysis and allows these enzymes to operate in a highly efficient and reversible manner.

[FeFe] hydrogenases are very active enzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. Their active site, the H-cluster, contains a unique binuclear iron complex, [2Fe] H , with CN - and CO ligands as well as an aza-propane-dithiolate (ADT) moiety featuring a central amine functionality that mediates proton transfer during catalysis. We present a pulsed 13 C-ENDOR investigation of the H-cluster in which the two methylene carbons of ADT are isotope labeled with 13 C. We observed that the corresponding two 13 C hyperfine interactions are of opposite sign and corroborated this finding using density functional theory calculations. The spin polarization in the ADT ligand is shown to be linked to the asymmetric coordination of the distal iron site with its terminal CN - and CO ligands. We propose that this asymmetry is relevant for the enzyme reactivity and is related to the (optimal) stabilization of the iron-hydride intermediate in the catalytic cycle.

Hydrogenases are metalloenzymes that catalyze the reversible oxidation of H 2 . The [FeFe] hydrogenases are generally biased toward proton reduction and have high activities. Several different catalytic mechanisms have been proposed for the [FeFe] enzymes based on the identification of intermediate states in equilibrium and steady state experiments. Here, we examine the kinetic competency of these intermediate states in the [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1), using a laser-induced potential jump and time-resolved IR (TRIR) spectroscopy. A CdSe/CdS dot-in-rod (DIR) nanocrystalline semiconductor is employed as the photosensitizer and a redox mediator efficiently transfers electrons to the enzyme. A pulsed laser induces a potential jump, and TRIR spectroscopy is used to follow the population flux through each intermediate state. The results clearly establish the kinetic competency of all intermediate populations examined: H ox , H red , H red H + , H sred H + , and H hyd . Additionally, a new short-lived intermediate species with a CO peak at 1896 cm -1 was identified. These results establish a kinetics framework for understanding the catalytic mechanism of [FeFe] hydrogenases.

Photosynthetic complex I enables cyclic electron flow around photosystem I, a regulatory mechanism for photosynthetic energy conversion. We report a 3.3-angstrom-resolution cryo-electron microscopy structure of photosynthetic complex I from the cyanobacterium Thermosynechococcus elongatus. The model reveals structural adaptations that facilitate binding and electron transfer from the photosynthetic electron carrier ferredoxin. By mimicking cyclic electron flow with isolated components in vitro, we demonstrate that ferredoxin directly mediates electron transfer between photosystem I and complex I, instead of using intermediates such as NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate). A large rate constant for association of ferredoxin to complex I indicates efficient recognition, with the protein subunit NdhS being the key component in this process., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

[FeFe] hydrogenases interconvert H 2 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 H 2 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 H 2 production activity but giving significantly higher H 2 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.

In this work we present a viologen-modified electrode providing protection for hydrogenases against high potential inactivation. Hydrogenases, including O2-tolerant classes, suffer from reversible inactivation upon applying high potentials, which limits their use in biofuel cells to certain conditions. Our previously reported protection strategy based on the integration of hydrogenase into redox matrices enabled the use of these biocatalysts in biofuel cells even under anode limiting conditions. However, mediated catalysis required application of an overpotential to drive the reaction, and this translates into a power loss in a biofuel cell. In the present work, the enzyme is adsorbed on top of a covalently-attached viologen layer which leads to mixed, direct and mediated, electron transfer processes; at low overpotentials, the direct electron transfer process generates a catalytic current, while the mediated electron transfer through the viologens at higher potentials generates a redox buffer that prevents oxidative inactivation of the enzyme. Consequently, the enzyme starts the catalysis at no overpotential with viologen self-activated protection at high potentials.

[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 H ox air . The formation of the H ox air 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 H ox air state in DdHydAB and the [FeFe] hydrogenase from Chlamydomonas reinhardtii, which is based on high potential inactivation in the presence of sulfide.

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H 2 level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from Thermotoga maritima (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the H ox state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the H ox state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster.

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (H hyd ) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57 Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, and the Fe-H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that H hyd is the catalytic state one step prior to H 2 formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H 2 bond formation by [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 pK a of its one electron reduced state. This may help to sustain H 2 production at high pH values. These results may apply to all [FeFe] hydrogenases containing accessory clusters.

[FeFe] hydrogenases are extremely active and efficient H 2 -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 H 2 -converting catalysts.

The applicability of the extremely active [FeFe] hydrogenase from Desulfovibrio desulfuricans in H 2 -producing devices is studied. Despite being the most active enzyme for H 2 catalysis, its high sensitivity towards O 2 has prevented its use in electrolytic water splitting cells. Using electrochemical methods, the catalytic activity of the enzyme at H 2 -producing potentials and its inactivation upon exposure to limited amounts of O 2 or under illumination is analysed. This enzyme is shown to maintain H 2 production activity for extended periods of time at low potentials. At such potentials, the enzyme can deliver the required electrons to fully reduce O 2 to H 2 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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN - ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S] H 2+ ⇔ [4Fe-4S] H + and [Fe(I)Fe(II)] H ⇔ [Fe(I)Fe(I)] H thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H + + 2e - ⇄ H 2 . Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state H red of the H-cluster actually consists of two species: H red = [4Fe-4S] H + - [Fe(I)Fe(II)] H and H red H + = [4Fe-4S] H 2+ - [Fe(I)Fe(I)] H (H + ) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S] H subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (H red /H red H + ) has a pK a ≈ 7.2.

Iron-sulfur clusters form one of the largest and most diverse classes of enzyme cofactors in nature. They may serve as structural factors, form electron transfer chains between active sites and external redox partners, or form components of enzyme active sites. Their specific role is a consequence of the cluster type and the surrounding protein environment. The relative effects of these factors are not completely understood, and it is not yet possible to predict the properties of iron-sulfur clusters based on amino acid sequences or rationally tune their properties to generate proteins with new desirable functions. Here, we generate mutations in a [2Fe-2S] cluster protein, the TmHydC subunit of the trimeric [FeFe]-hydrogenase from Thermotoga maritima, to study the factors that affect its redox potential. Saturation mutagenesis of Val131 was used to tune the redox potential over a 135 mV range and revealed that cluster redox potential and electronic properties correlate with amino acid hydrophobicity and the ability to form hydrogen bonds to the cluster. Proline scanning mutagenesis between pairs of ligating cysteines was used to remove backbone amide hydrogen bonds to the cluster and decrease the redox potential by up to 132 mV, without large structural changes in most cases. However, substitution of Gly83 with proline caused a change of HydC to a [4Fe-4S] cluster protein with a redox potential of -526 mV. Together, these results confirm the importance of hydrogen bonding in tuning cluster redox potentials and demonstrate the versatility of iron-sulfur cluster protein folds at binding different types of clusters.

NADH:quinone oxidoreductase (complex I) couples NADH oxidation and quinone reduction to proton translocation across an energy-transducing membrane. All complexes I contain a flavin to oxidize NADH, seven iron-sulfur clusters to transfer electrons from the flavin to quinone and an eighth cluster (N1a) on the opposite side of the flavin. The role of cluster N1a is unknown, but Escherichia coli complex I has an unusually high-potential cluster N1a and its reduced flavin produces H2O2, not superoxide, suggesting that cluster N1a may affect reactive oxygen species production. In the present study, we combine protein film voltammetry with mutagenesis in overproduced N1a-binding subunits to identify two residues that switch N1a between its high- (E. coli, valine and asparagine) and low- (Bos taurus and Yarrowia lipolytica, proline and methionine) potential forms. The mutations were incorporated into E. coli complex I: cluster N1a could no longer be reduced by NADH, but H2O2 and superoxide production were unaffected. The reverse mutations (that increase the potential by ~0.16 V) were incorporated into Y. lipolytica complex I, but N1a was still not reduced by NADH. We conclude that cluster N1a does not affect reactive oxygen species production by the complex I flavin; it is probably required for enzyme assembly or stability.

A highly enantioselective addition of phenyl carbamate to meso-epoxides has been developed to efficiently generate protected trans-1,2-amino alcohols. This transformation is promoted by an oligomeric (salen)Co-OTf catalyst and has been used to prepare two useful 2-aminocycloalkanol hydrochlorides in enantiopure form on a multigram scale from commercially available starting materials.

NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. During catalysis, NADH oxidation by a flavin mononucleotide is followed by electron transfer to a chain of iron-sulfur clusters. Alternatively, the flavin may be reoxidized by hydrophilic electron acceptors, by artificial electron acceptors in kinetic studies, or by oxygen and redox-cycling molecules to produce reactive oxygen species. Here, we study two steps in the mechanism of NADH oxidation by complex I. First, molecular fragments of NAD(H), tested as flavin-site inhibitors or substrates, reveal that the adenosine moiety is crucial for binding. Nicotinamide-containing fragments that lack the adenosine do not bind, and ADP-ribose binds more strongly than NAD(+), suggesting that the nicotinamide is detrimental to binding. Second, the primary kinetic isotope effects from deuterated nicotinamide nucleotides confirm that hydride transfer is from the pro-S position and reveal that hydride transfer, along with NAD(+) dissociation, is partially rate-limiting. Thus, the transition state energies are balanced so that no single step in NADH oxidation is completely rate-limiting. Only at very low NADH concentrations does weak NADH binding limit NADH:ubiquinone oxidoreduction, and at the high nucleotide concentrations of the mitochondrial matrix, weak nucleotide binding constants assist product dissociation. Using fast nucleotide reactions and a balance between the nucleotide binding constants and concentrations, complex I combines fast and energy-conserving NADH oxidation with minimal superoxide production from the nucleotide-free site.

A highly enantioselective acylation of silyl ketene acetals with acyl fluorides has been developed to generate useful α,α-disubstituted butyrolactone products. This transformation is promoted by a new thiourea catalyst and 4-pyrrolidinopyridine and represents the first example of enantioselective thiourea anion-binding catalysis with fluoride.

The flavin mononucleotide in complex I (NADH:ubiquinone oxidoreductase) catalyzes NADH oxidation, O(2) reduction to superoxide, and the reduction of several 'artificial' electron acceptors. Here, we show that the positively-charged electron acceptors paraquat and hexaammineruthenium(III) react with the nucleotide-bound reduced flavin in complex I, by an unusual ternary mechanism. NADH, ATP, ADP and ADP-ribose stimulate the reactions, indicating that the positively-charged acceptors interact with their negatively-charged phosphates. Our mechanism for paraquat reduction defines a new mechanism for superoxide production by complex I (by redox cycling); in contrast to direct O(2) reduction the rate is stimulated, not inhibited, by high NADH concentrations., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Complex I (NADH:ubiquinone oxidoreductase) is crucial to respiration in many aerobic organisms. The hydrophilic domain of complex I, containing nine or more redox cofactors, and comprising seven conserved core subunits, protrudes into the mitochondrial matrix or bacterial cytoplasm. The α-helical membrane-bound hydrophobic domain contains a further seven core subunits that are mitochondrial-encoded in eukaryotes and named the ND subunits (ND1-ND6 and ND4L). Complex I couples the oxidation of NADH in the hydrophilic domain to ubiquinone reduction and proton translocation in the hydrophobic domain. Although the mechanisms of NADH oxidation and intramolecular electron transfer are increasingly well understood, the mechanisms of ubiquinone reduction and proton translocation remain only poorly defined. Recently, an α-helical model of the hydrophobic domain of bacterial complex I [Efremov, Baradaran and Sazanov (2010) Nature 465, 441-447] revealed how the 63 transmembrane helices of the seven core subunits are arranged, and thus laid a foundation for the interpretation of functional data and the formulation of mechanistic proposals. In the present paper, we aim to correlate information from sequence analyses, site-directed mutagenesis studies and mutations that have been linked to human diseases, with information from the recent structural model. Thus we aim to identify and discuss residues in the ND subunits of mammalian complex I which are important in catalysis and for maintaining the enzyme's structural and functional integrity.

Three of the conserved, membrane-bound subunits in NADH:ubiquinone oxidoreductase (complex I) are related to one another, and to Mrp sodium-proton antiporters. Recent structural analysis of two prokaryotic complexes I revealed that the three subunits each contain fourteen transmembrane helices that overlay in structural alignments: the translocation of three protons may be coordinated by a lateral helix connecting them together (Efremov, R.G., Baradaran, R. and Sazanov, L.A. (2010). The architecture of respiratory complex I. Nature 465, 441-447). Here, we show that in higher metazoans the threefold symmetry is broken by the loss of three helices from subunit ND2; possible implications for the mechanism of proton translocation are discussed., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory chain enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. Alternatively, NADH oxidation, by the flavin mononucleotide in complex I, can be coupled to the reduction of hydrophilic electron acceptors, in non-energy-transducing reactions. The reduction of molecular oxygen and hydrophilic quinones leads to the production of reactive oxygen species, the reduction of nicotinamide nucleotides leads to transhydrogenation, and "artificial" electron acceptors are widely used to study the mechanism of NADH oxidation. Here, we use a combined modeling strategy to accurately describe data from three flavin-linked electron acceptors (molecular oxygen, APAD(+), and ferricyanide), in the presence and absence of a competitive inhibitor, ADP-ribose. Our combined ping-pong (or ping-pong-pong) mechanism comprises the Michaelis-Menten equation for the reactions of NADH and APAD(+), simple dissociation constants for nonproductive nucleotide-enzyme complexes (defined for specific flavin oxidation states), and second-order rate constants for the reactions of ferricyanide and oxygen. The NADH-dependent parameters are independent of the identity of the electron acceptor. In contrast, a further flavin-linked acceptor, hexaammineruthenium(III), does not obey ping-pong-pong kinetics, and alternative sites for its reaction are discussed. Our analysis provides kinetic and thermodynamic information about the reactions of the flavin active site in complex I that is relevant to understanding the physiologically relevant mechanisms of NADH oxidation and superoxide formation.