Replacing a Cysteine Ligand by Selenocysteine in a [NiFe]-Hydrogenase Unlocks Hydrogen Production Activity and Addresses the Role of Concerted Proton-Coupled Electron Transfer in Electrocatalytic Reversibility.

Hydrogenases catalyze hydrogen/proton interconversion that is normally electrochemically reversible (having minimal overpotential requirement), a special property otherwise almost exclusive to platinum metals. The mechanism of [NiFe]-hydrogenases includes a long-range proton-coupled electron-transfer process involving a specific Ni-coordinated cysteine and the carboxylate of a nearby glutamate. A variant in which this cysteine has been exchanged for selenocysteine displays two distinct changes in electrocatalytic properties, as determined by protein film voltammetry. First, proton reduction, even in the presence of H2 (a strong product inhibitor), is greatly enhanced relative to H2 oxidation: this result parallels a characteristic of natural [NiFeSe]-hydrogenases which are superior H2 production catalysts. Second, an inflection (an S-shaped "twist" in the trace) appears around the formal potential, the small overpotentials introduced in each direction (oxidation and reduction) signaling a departure from electrocatalytic reversibility. Concerted proton-electron transfer offers a lower energy pathway compared to stepwise transfers. Given the much lower proton affinity of Se compared to that of S, the inflection provides compelling evidence that concerted proton-electron transfer is important in determining why [NiFe]-hydrogenases are reversible electrocatalysts.

Controlled Biocatalytic Synthesis of a Metal Nanoparticle-Enzyme Hybrid: Demonstration for Catalytic H2-driven NADH Recycling.

Here we demonstrate the preparation of enzyme-metal biohybrids of NAD+ reductase with biocatalytically-synthesised small gold nanoparticles (NPs, <10 nm) and core-shell gold-platinum NPs for tandem catalysis. Despite the variety of methods available for NP synthesis, there remains a need for more sustainable strategies which also give precise control over the shape and size of the metal NPs for applications in catalysis, biomedical devices, and electronics. We demonstrate facile biosynthesis of spherical, highly uniform, gold NPs under mild conditions using an isolated enzyme moiety, an NAD+ reductase, to reduce metal salts while oxidising a nicotinamide-containing cofactor. By subsequently introducing platinum salts, we show that core-shell Au@Pt NPs can then be formed. Catalytic function of these enzyme-Au@Pt NP hybrids was demonstrated for H2-driven NADH recycling to support enantioselective ketone reduction by an NADH-dependent alcohol dehydrogenase.

Biocatalytic reductive amination as a route to isotopically labelled amino acids suitable for analysis of large proteins by NMR.

We demonstrate an atom-efficient and easy to use H2-driven biocatalytic platform for the enantioselective incorporation of 2H-atoms into amino acids. By combining the biocatalytic deuteration catalyst with amino acid dehydrogenase enzymes capable of reductive amination, we synthesised a library of multiply isotopically labelled amino acids from low-cost isotopic precursors, such as 2H2O and 15NH4+. The chosen approach avoids the use of pre-labeled 2H-reducing agents, and therefore vastly simplifies product cleanup. Notably, this strategy enables 2H, 15N, and an asymmetric centre to be introduced at a molecular site in a single step, with full selectivity, under benign conditions, and with near 100% atom economy. The method facilitates the preparation of amino acid isotopologues on a half-gram scale. These amino acids have wide applicability in the analytical life sciences, and in particular for NMR spectroscopic analysis of proteins. To demonstrate the benefits of the approach for enabling the workflow of protein NMR chemists, we prepared l-[α-2H,15N, ß-13C]-alanine and integrated it into a large (>400 kDa) heat-shock protein oligomer, which was subsequently analysable by methyl-TROSY techniques, revealing new structural information.

Developing high-affinity, oxygen-insensitive [NiFe]-hydrogenases as biocatalysts for energy conversion.

The splitting of hydrogen (H2) is an energy-yielding process, which is important for both biological systems and as a means of providing green energy. In biology, this reaction is mediated by enzymes called hydrogenases, which utilise complex nickel and iron cofactors to split H2 and transfer the resulting electrons to an electron-acceptor. These [NiFe]-hydrogenases have received considerable attention as catalysts in fuel cells, which utilise H2 to produce electrical current. [NiFe]-hydrogenases are a promising alternative to the platinum-based catalysts that currently predominate in fuel cells due to the abundance of nickel and iron, and the resistance of some family members to inhibition by gases, including carbon monoxide, which rapidly poison platinum-based catalysts. However, the majority of characterised [NiFe]-hydrogenases are inhibited by oxygen (O2), limiting their activity and stability. We recently reported the isolation and characterisation of the [NiFe]-hydrogenase Huc from Mycobacterium smegmatis, which is insensitive to inhibition by O2 and has an extremely high affinity, making it capable of oxidising H2 in air to below atmospheric concentrations. These properties make Huc a promising candidate for the development of enzyme-based fuel cells (EBFCs), which utilise H2 at low concentrations and in impure gas mixtures. In this review, we aim to provide context for the use of Huc for this purpose by discussing the advantages of [NiFe]-hydrogenases as catalysts and their deployment in fuel cells. We also address the challenges associated with using [NiFe]-hydrogenases for this purpose, and how these might be overcome to develop EBFCs that can be deployed at scale.

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.

Electrochemical experiments define potentials associated with binding of substrates and inhibitors to nitrogenase MoFe protein.

Nitrogenases catalyse the 6-electron reduction of dinitrogen to ammonia, passing through a series of redox and protonation levels during catalytic substrate reduction. The molybdenum-iron nitrogenase is the most well-studied, but redox potentials associated with proton-coupled transformations between the redox levels of the catalytic MoFe protein have proved difficult to pin down, in part due to a complex electron-transfer pathway from the partner Fe protein, linked to ATP-hydrolysis. Here, we apply electrochemical control to the MoFe protein of Azotobacter vinelandii nitrogenase, using europium(III/II)-ligand couples as low potential redox mediators. We combine insight from the electrochemical current response with data from gas chromatography and in situ infrared spectroscopy, in order to define potentials for the binding of a series of inhibitors (carbon monoxide, methyl isocyanide) to the metallo-catalytic site of the MoFe protein, and the onset of catalytic transformation of alternative substrates (protons and acetylene) by the enzyme. Thus, we associate potentials with the redox levels for inhibition and catalysis by nitrogenase, with relevance to the elusive mechanism of biological nitrogen fixation.

Structural basis for bacterial energy extraction from atmospheric hydrogen.

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air.

A pharmacophore-based approach to demonstrating the scope of alcohol dehydrogenases.

Barriers to the ready adoption of biocatalysis into asymmetric synthesis for early stage medicinal chemistry are addressed, using ketone reduction by alcohol dehydrogenase as a model reaction. An efficient substrate screening approach is used to show the wide substrate scope of commercial alcohol dehydrogenase enzymes, with a high tolerance to chemical groups employed in drug discovery (heterocycle, trifluoromethyl and nitrile/nitro groups) observed. We use our screening data to build a preliminary predictive pharmacophore-based screening tool using Forge software, with a precision of 0.67/1, demonstrating the potential for developing substrate screening tools for commercially available enzymes without publicly available structures. We hope that this work will facilitate a culture shift towards adopting biocatalysis alongside traditional chemical catalytic methods in early stage drug discovery.

A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H.

We demonstrate a recycling system for synthetic nicotinamide cofactor analogues using a soluble hydrogenase with turnover number of >1000 for reduction of the cofactor analogues by H2. Coupling this system to an ene reductase, we show quantitative conversion of N-ethylmaleimide to N-ethylsuccinimide. The biocatalyst system retained >50% activity after 7 h.

The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase.

Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure-function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1 (Hyd1) from Escherichia coli makes it possible to navigate through the full array of active site states previously observed in solution. Electrochemical control is combined with synchrotron infrared microspectroscopy, which enables us to measure high signal-to-noise IR spectra in situ from a small area of crystal. The output reports on active site speciation via the vibrational stretching band positions of the endogenous CO and CN- ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle.

Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site.

Elucidating the distribution of intermediates at the active site of redox metalloenzymes is vital to understanding their highly efficient catalysis. Here we demonstrate that it is possible to generate, and detect, the key catalytic redox states of an [FeFe]-hydrogenase in a protein crystal. Individual crystals of the prototypical [FeFe]-hydrogenase I from Clostridium pasteurianum (CpI) are maintained under electrochemical control, allowing for precise tuning of the redox potential, while the crystal is simultaneously probed via Fourier Transform Infrared (FTIR) microspectroscopy. The high signal/noise spectra reveal potential-dependent variation in the distribution of redox states at the active site (H-cluster) according to state-specific vibrational bands from the endogeneous CO and CN- ligands. CpI crystals are shown to populate the same H-cluster states as those detected in solution, including the oxidised species Hox, the reduced species Hred/HredH+, the super-reduced HsredH+ and the hydride species Hhyd. The high sensitivity and precise redox control offered by this approach also facilitates the detection and characterisation of low abundance species that only accumulate within a narrow window of conditions, revealing new redox intermediates.

Chemo-bio catalysis using carbon supports: application in H2-driven cofactor recycling.

Heterogeneous biocatalytic hydrogenation is an attractive strategy for clean, enantioselective C[double bond, length as m-dash]X reduction. This approach relies on enzymes powered by H2-driven NADH recycling. Commercially available carbon-supported metal (metal/C) catalysts are investigated here for direct H2-driven NAD+ reduction. Selected metal/C catalysts are then used for H2 oxidation with electrons transferred via the conductive carbon support material to an adsorbed enzyme for NAD+ reduction. These chemo-bio catalysts show improved activity and selectivity for generating bioactive NADH under ambient reaction conditions compared to metal/C catalysts. The metal/C catalysts and carbon support materials (all activated carbon or carbon black) are characterised to probe which properties potentially influence catalyst activity. The optimised chemo-bio catalysts are then used to supply NADH to an alcohol dehydrogenase for enantioselective (>99% ee) ketone reductions, leading to high cofactor turnover numbers and Pd and NAD+ reductase activities of 441 h-1 and 2347 h-1, respectively. This method demonstrates a new way of combining chemo- and biocatalysis on carbon supports, highlighted here for selective hydrogenation reactions.

Hybrid Chemo-, Bio-, and Electrocatalysis for Atom-Efficient Deuteration of Cofactors in Heavy Water.

Deuterium-labeled nicotinamide cofactors such as [4-2H]-NADH can be used as mechanistic probes in biological redox processes and offer a route to the synthesis of selectively [2H] labeled chemicals via biocatalytic reductive deuteration. Atom-efficient routes to the formation and recycling of [4-2H]-NADH are therefore highly desirable but require careful design in order to alleviate the requirement for [2H]-labeled reducing agents. In this work, we explore a suite of electrode or hydrogen gas driven catalyst systems for the generation of [4-2H]-NADH and consider their use for driving reductive deuteration reactions. Catalysts are evaluated for their chemoselectivity, stereoselectivity, and isotopic selectivity, and it is shown that inclusion of an electronically coupled NAD+-reducing enzyme delivers considerable advantages over purely metal based systems, yielding exclusively [4S-2H]-NADH. We further demonstrate the applicability of these types of [4S-2H]-NADH recycling systems for driving reductive deuteration reactions, regardless of the facioselectivity of the coupled enzyme.

E. coli Nickel-Iron Hydrogenase 1 Catalyses Non-native Reduction of Flavins: Demonstration for Alkene Hydrogenation by Old Yellow Enzyme Ene-reductases*.

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25-70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "ene-reductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min-1 and total turnover numbers up to 20 200 were recorded during flavin recycling.

Synthesis of [4S-2 H]NADH, [4R-2 H]NADH, [4-2 H2 ]NADH and [4-2 H]NAD+ cofactors through heterogeneous biocatalysis in heavy water.

This practitioner protocol describes the synthesis of a family of deuterated nicotinamide cofactors: [4S-2 H]NADH, [4R-2 H]NADH, [4-2 H2 ]NADH and [4-2 H]NAD+ . The application of a recently developed H2 -driven heterogeneous biocatalyst enables the cofactors to be prepared with high (>90%) 2 H-incorporation with 2 H2 O as the only isotope source.

E. coli Nickel-Iron Hydrogenase 1 Catalyses Non-native Reduction of Flavins: Demonstration for Alkene Hydrogenation by Old Yellow Enzyme Ene-reductases.

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25-70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "ene-reductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min-1 and total turnover numbers up to 20 200 were recorded during flavin recycling.