Outer-sphere effects on the O2 sensitivity, catalytic bias and catalytic reversibility of hydrogenases.

The comparison of homologous metalloenzymes, in which the same inorganic active site is surrounded by a variable protein matrix, has demonstrated that residues that are remote from the active site may have a great influence on catalytic properties. In this review, we summarise recent findings on the diverse molecular mechanisms by which the protein matrix may define the oxygen tolerance, catalytic directionality and catalytic reversibility of hydrogenases, enzymes that catalyse the oxidation and evolution of H2. These mechanisms involve residues in the second coordination sphere of the active site metal ion, more distant residues affecting protein flexibility through their side chains, residues lining the gas channel and even accessory subunits. Such long-distance effects, which contribute to making enzymes efficient, robust and different from one another, are a source of wonder for biochemists and a challenge for synthetic bioinorganic chemists.

Kinetic Modeling of the Reversible or Irreversible Electrochemical Responses of FeFe-Hydrogenases.

The enzyme FeFe-hydrogenase catalyzes H2 evolution and oxidation at an active site that consists of a [4Fe-4S] cluster bridged to a [Fe2(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 H2 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.

An allosteric redox switch involved in oxygen protection in a CO2 reductase.

Metal-dependent formate dehydrogenases reduce CO2 with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O2 exposure.

A Chimeric NiFe Hydrogenase Heterodimer to Assess the Role of the Electron Transfer Chain in Tuning the Enzyme's Catalytic Bias and Oxygen Tolerance.

The observation that some homologous enzymes have the same active site but very different catalytic properties demonstrates the importance of long-range effects in enzyme catalysis, but these effects are often difficult to rationalize. The NiFe hydrogenases 1 and 2 (Hyd 1 and Hyd 2) from E. coli both consist of a large catalytic subunit that embeds the same dinuclear active site and a small electron-transfer subunit with a chain of three FeS clusters. Hyd 1 is mostly active in H2 oxidation and resistant to inhibitors, whereas Hyd 2 also catalyzes H2 production and is strongly inhibited by O2 and CO. Based on structural and site-directed mutagenesis data, it is currently believed that the catalytic bias and tolerance to O2 of Hyd 1 are defined by the distal and proximal FeS clusters, respectively. To test these hypotheses, we produced and characterized a hybrid enzyme made of the catalytic subunit of Hyd 1 and the electron transfer subunit of Hyd 2. We conclude that catalytic bias and sensitivity to CO are set by the catalytic subunit rather than by the electron transfer chain. We confirm the importance of the proximal cluster in making the enzyme Hyd 1 resist long-term exposure to O2, but we show that other structural determinants, in both subunits, contribute to O2 tolerance. A similar strategy based on the design of chimeric heterodimers could be used in the future to elucidate various structure-function relationships in hydrogenases and other multimeric metalloenzymes and to engineer useful hydrogenases that combine the desirable properties of distinct, homologous enzymes.

Light-Driven Hydrogen Evolution Reaction Catalyzed by a Molybdenum-Copper Artificial Hydrogenase.

Orange protein (Orp) is a small bacterial metalloprotein of unknown function that harbors a unique molybdenum/copper (Mo/Cu) heterometallic cluster, [S2MoS2CuS2MoS2]3-. In this paper, the performance of Orp as a catalyst for the photocatalytic reduction of protons into H2 has been investigated under visible light irradiation. We report the complete biochemical and spectroscopic characterization of holo-Orp containing the [S2MoS2CuS2MoS2]3- cluster, with docking and molecular dynamics simulations suggesting a positively charged Arg, Lys-containing pocket as the binding site. Holo-Orp exhibits excellent photocatalytic activity, in the presence of ascorbate as the sacrificial electron donor and [Ru(bpy)3]Cl2 as the photosensitizer, for hydrogen evolution with a maximum turnover number of 890 after 4 h irradiation. Density functional theory (DFT) calculations were used to propose a consistent reaction mechanism in which the terminal sulfur atoms are playing a key role in promoting H2 formation. A series of dinuclear [S2MS2M'S2MS2](4n)- clusters, with M = MoVI, WVI and M'(n+) = CuI, FeI, NiI, CoI, ZnII, CdII were assembled in Orp, leading to different M/M'-Orp versions which are shown to display catalytic activity, with the Mo/Fe-Orp catalyst giving a remarkable turnover number (TON) of 1150 after 2.5 h reaction and an initial turnover frequency (TOF°) of 800 h-1 establishing a record among previously reported artificial hydrogenases.

Transport limited adsorption experiments give a new lower estimate of the turnover frequency of Escherichia coli hydrogenase 1.

Protein Film Electrochemistry is a technique in which a redox enzyme is directly wired to an electrode, which substitutes for the natural redox partner. In this technique, the electrical current flowing through the electrode is proportional to the catalytic activity of the enzyme. However, in most cases, the amount of enzyme molecules contributing to the current is unknown and the absolute turnover frequency cannot be determined. Here, we observe the formation of electrocatalytically active films of E. coli hydrogenase 1 by rotating an electrode in a sub-nanomolar solution of enzyme. This process is slow, and we show that it is mass-transport limited. Measuring the rate of the immobilization allows the determination of an estimation of the turnover rate of the enzyme, which appears to be much greater than that deduced from solution assays under the same conditions.

Increasing the O2 Resistance of the [FeFe]-Hydrogenase CbA5H through Enhanced Protein Flexibility.

The high turnover rates of [FeFe]-hydrogenases under mild conditions and at low overpotentials provide a natural blueprint for the design of hydrogen catalysts. However, the unique active site (H-cluster) degrades upon contact with oxygen. The [FeFe]-hydrogenase fromClostridium beijerinckii (CbA5H) is characterized by the flexibility of its protein structure, which allows a conserved cysteine to coordinate to the active site under oxidative conditions. Thereby, intrinsic cofactor degradation induced by dioxygen is minimized. However, the protection from O2 is only partial, and the activity of the enzyme decreases upon each exposure to O2. By using site-directed mutagenesis in combination with electrochemistry, ATR-FTIR spectroscopy, and molecular dynamics simulations, we show that the kinetics of the conversion between the oxygen-protected inactive state (cysteine-bound) and the oxygen-sensitive active state can be accelerated by replacing a surface residue that is very distant from the active site. This sole exchange of methionine for a glutamate residue leads to an increased resistance of the hydrogenase to dioxygen. With our study, we aim to understand how local modifications of the protein structure can have a crucial impact on protein dynamics and how they can control the reactivity of inorganic active sites through outer sphere effects.

Assessing the Extent of Potential Inversion by Cyclic Voltammetry: Theory, Pitfalls, and Application to a Nickel Complex with Redox-Active Iminosemiquinone Ligands.

Potential inversion refers to the situation where a protein cofactor or a synthetic molecule can be oxidized or reduced twice in a cooperative manner; that is, the second electron transfer is easier than the first. This property is very important regarding the catalytic mechanism of enzymes that bifurcate electrons and the properties of bidirectional redox molecular catalysts that function in either direction of the reaction with no overpotential. Cyclic voltammetry is the most common technique for characterizing the thermodynamics and kinetics of electron transfer to or from these molecules. However, a gap in the literature is the absence of analytical predictions to help interpret the values of the voltammetric peak potentials when potential inversion occurs; the cyclic voltammograms are therefore often analyzed by simulating the data, with no discussion of the possibility of overfitting and often no estimation of the error on the determined parameters. Here we formulate the theory for the voltammetry of freely diffusing or surface-confined two-electron redox species in the experimentally relevant irreversible limit where the peak separation depends on the scan rate. We explain why the model is intrinsically underdetermined, and we illustrate this conclusion by analysis of the voltammetry of a nickel complex with redox-active iminosemiquinone ligands. Being able to characterize the thermodynamics of two-electron electron-transfer reactions will be crucial for designing more efficient catalysts.

Electrochemical Kinetics Support a Second Coordination Sphere Mechanism in Metal-Based Formate Dehydrogenase.

Metal-based formate dehydrogenases are molybdenum or tungsten-dependent enzymes that catalyze the interconversion between formate and CO2 . According to the current consensus, the metal ion of the catalytic center in its active form is coordinated by 6 S (or 5 S and 1 Se) atoms, leaving no free coordination sites to which formate could bind to the metal. Some authors have proposed that one of the active site ligands decoordinates during turnover to allow formate binding. Another proposal is that the oxidation of formate takes place in the second coordination sphere of the metal. Here, we have used electrochemical steady-state kinetics to elucidate the order of the steps in the catalytic cycle of two formate dehydrogenases. Our results strongly support the "second coordination sphere" hypothesis.

Second and Outer Coordination Sphere Effects in Nitrogenase, Hydrogenase, Formate Dehydrogenase, and CO Dehydrogenase.

Gases like H2, N2, CO2, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N2, CO2, and CO and the production of H2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N2 fixation by nitrogenase and H2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere.

Reversible or Irreversible Catalysis of H+/H2 Conversion by FeFe Hydrogenases.

Studies of molecular catalysts traditionally aim at understanding how a certain mechanism allows the reaction to be fast. A distinct question, which has only recently received attention in the case of bidirectional molecular catalysts, is how much thermodynamic driving force is required to achieve fast catalysis in either direction of the reaction. "Reversible" catalysts are bidirectional catalysts that work either way in response to even a small departure from equilibrium and thus do not waste input free energy as heat; conversely, "irreversible" catalysts require a large driving force to proceed at an appreciable rate [Fourmond et al. Nat. Rev. Chem. 2021, 5, 348-360]. Numerous mechanistic rationales for these contrasting behaviors have been proposed. To understand the determinants of catalytic (ir)reversibility, we examined the steady-state, direct electron transfer voltammetry of a particular FeFe hydrogenase, from Thermoanaerobacter mathranii, which is very unusual in that it irreversibly catalyzes H2 oxidation and production: a large overpotential is required for the reaction to proceed in either direction [Land et al. Chem. Sci. 2020, 11, 12789-12801]. In contrast to previous hypotheses, we demonstrate that in this particular enzyme catalytic irreversibility can be explained without invoking slow interfacial electron transfer or variations in the mechanism: the observed kinetics is fully consistent with the same catalytic pathway being used in both directions of the reaction.

Electrochemical Studies of CO2 -Reducing Metalloenzymes.

Only two enzymes are capable of directly reducing CO2 : CO dehydrogenase, which produces CO at a [NiFe4 S4 ] active site, and formate dehydrogenase, which produces formate at a mononuclear W or Mo active site. Both metalloenzymes are very rapid, energy-efficient and specific in terms of product. They have been connected to electrodes with two different objectives. A series of studies used protein film electrochemistry to learn about different aspects of the mechanism of these enzymes (reactivity with substrates, inhibitors…). Another series focused on taking advantage of the catalytic performance of these enzymes to build biotechnological devices, from CO2 -reducing electrodes to full photochemical devices performing artificial photosynthesis. Here, we review all these works.

Reversible H2 Oxidation and Evolution by Hydrogenase Embedded in a Redox Polymer Film.

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 H2/O2 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 H2 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.

A safety cap protects hydrogenase from oxygen attack.

[FeFe]-hydrogenases are efficient H2-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic Hox-state and the inactive but oxygen-resistant Hinact-state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O2-binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 Å away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues.

Formate Dehydrogenases Reduce CO2 Rather than HCO3- : An Electrochemical Demonstration.

Mo/W formate dehydrogenases catalyze the reversible reduction of CO2 species to formate. It is thought that the substrate is CO2 and not a hydrated species like HCO3- , but there is still no indisputable evidence for this, in spite of the extreme importance of the nature of the substrate for mechanistic studies. We devised a simple electrochemical method to definitively demonstrate that the substrate of formate dehydrogenases is indeed CO2 .

Artificial maturation of [FeFe] hydrogenase in a redox polymer film.

We demonstrate that the insertion of the dinuclear active site of [FeFe] hydrogenase into the apo-enzyme can occur when the enzyme is embedded in a film of redox polymer, under conditions of mediated electron transfer. The maturation can be monitored by electrochemistry, and is as fast as under conditions of direct electron transfer. This new approach further clears the way to the implementation of hydrogenases in large scale real life processes.

Reversible catalysis.

We describe as 'reversible' a bidirectional catalyst that allows a reaction to proceed at a significant rate in response to even a small departure from equilibrium, resulting in fast and energy-efficient chemical transformation. Examining the relation between reaction rate and thermodynamic driving force is the basis of electrochemical investigations of redox reactions, which can be catalysed by metallic surfaces and biological or synthetic molecular catalysts. This relation has also been discussed in the context of biological energy transduction, regarding the function of biological molecular machines that harness chemical reactions to do mechanical work. This Perspective describes mean-field kinetic modelling of these three types of systems - surface catalysts, molecular catalysts of redox reactions and molecular machines - with the goal of unifying concepts in these different fields. We emphasize that reversibility should be distinguished from other figures of merit, such as rate or directionality, before its design principles can be identified and used to engineer synthetic catalysts.

The Solvent-Exposed Fe-S D-Cluster Contributes to Oxygen-Resistance in Desulfovibrio vulgaris Ni-Fe Carbon Monoxide Dehydrogenase.

Ni-Fe CO-dehydrogenases (CODHs) catalyze the conversion between CO and CO2 using a chain of Fe-S clusters to mediate long-range electron transfer. One of these clusters, the D-cluster, is surface-exposed and serves to transfer electrons between CODH and external redox partners. These enzymes tend to be extremely O2-sensitive and are always manipulated under strictly anaerobic conditions. However, the CODH from Desulfovibrio vulgaris (Dv) appears unique: exposure to micromolar concentrations of O2 on the minutes-time scale only reversibly inhibits the enzyme, and full activity is recovered after reduction. Here, we examine whether this unusual property of Dv CODH results from the nature of its D-cluster, which is a [2Fe-2S] cluster, instead of the [4Fe-4S] cluster observed in all other characterized CODHs. To this aim, we produced and characterized a Dv CODH variant where the [2Fe-2S] D-cluster is replaced with a [4Fe-4S] D-cluster through mutagenesis of the D-cluster-binding sequence motif. We determined the crystal structure of this CODH variant to 1.83-Å resolution and confirmed the incorporation of a [4Fe-4S] D-cluster. We show that upon long-term O2-exposure, the [4Fe-4S] D-cluster degrades, whereas the [2Fe-2S] D-cluster remains intact. Crystal structures of the Dv CODH variant exposed to O2 for increasing periods of time provide snapshots of [4Fe-4S] D-cluster degradation. We further show that the WT enzyme purified under aerobic conditions retains 30% activity relative to a fully anaerobic purification, compared to 10% for the variant, and the WT enzyme loses activity more slowly than the variant upon prolonged aerobic storage. The D-cluster is therefore a key site of irreversible oxidative damage in Dv CODH, and the presence of a [2Fe-2S] D-cluster contributes to the O2-tolerance of this enzyme. Together, these results relate O2-sensitivity with the details of the protein structure in this family of enzymes.

The two CO-dehydrogenases of Thermococcus sp. AM4.

Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO2 as a source of carbon. The recent detailed characterizations of some of them have evidenced a great diversity in terms of catalytic properties and resistance to O2. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp. AM4 (Tc). We have also crystallized CooS2, which is coupled in vivo to a hydrogenase. CooS1 and CooS2 are homodimers, and harbour five metalloclusters: two [Ni4Fe-4S] C clusters, two [4Fe-4S] B clusters and one interfacial [4Fe-4S] D cluster. We show that both are dependent on a maturase, CooC1 or CooC2, which is interchangeable. The homologous protein CooC3 does not allow Ni insertion in either CooS. The two CODHs from Tc have similar properties: they can both oxidize and produce CO. The Michaelis constants (Km) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO2. Product inhibition is observed only for CO2 reduction, consistent with CO2 binding being much weaker than CO binding. The two enzymes are rather O2 sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O2 than any other CODH for which these data are available.