Exploiting hydrogenases for biocatalytic hydrogenations.

The ability of hydrogenase enzymes to activate H with excellent selectivity leads to many interesting possibilities for biotechnology driven by H as a clean reductant. Here, we review examples where hydrogenase enzymes have been used to drive native and non-native hydrogenation reactions in solution or as part of a redox cascade on a conductive support, with a focus on the developments we have contributed to this field. In all of the examples discussed, hydrogenation reactions are enabled by coupled redox reactions: the oxidation of H at a hydrogenase active site, linked electronically ( relay clusters in the enzyme and/or conductive support) to the site of a reduction reaction, and we note how this parallels developments in site-separated reactivity in heterogeneous catalysis.

Selective hydrogenation of nitro compounds to amines by coupled redox reactions over a heterogeneous biocatalyst.

Cleaner synthesis of amines remains a key challenge in organic chemistry because of their prevalence in pharmaceuticals, agrochemicals and synthetic building blocks. Here, we report a different paradigm for chemoselective hydrogenation of nitro compounds to amines, under mild, aqueous conditions. The hydrogenase enzyme releases electrons from H to a carbon black support which facilitates nitro-group reduction.

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.

Controlled Biocatalytic Synthesis of a Metal Nanoparticle-Enzyme Hybrid: Demonstration for Catalytic H-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.

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 H-driven biocatalytic platform for the enantioselective incorporation of H-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 HO and NH. The chosen approach avoids the use of pre-labeled H-reducing agents, and therefore vastly simplifies product cleanup.

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 .

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 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 H and reduced methyl viologen.

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 nitrogenase, using europium(III/II)-ligand couples as low potential redox mediators.

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.

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 H. Coupling this system to an ene reductase, we show quantitative conversion of -ethylmaleimide to -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 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 from a small area of crystal.

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 (CpI) are maintained under electrochemical control, allowing for precise tuning of the redox potential, while the crystal is simultaneously probed Fourier Transform Infrared (FTIR) microspectroscopy.

Chemo-bio catalysis using carbon supports: application in H-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 H-driven NADH recycling. Commercially available carbon-supported metal (metal/C) catalysts are investigated here for direct H-driven NAD reduction.

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 H 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.

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

Deuterium-labeled nicotinamide cofactors such as [4-H]-NADH can be used as mechanistic probes in biological redox processes and offer a route to the synthesis of selectively [H] labeled chemicals biocatalytic reductive deuteration. Atom-efficient routes to the formation and recycling of [4-H]-NADH are therefore highly desirable but require careful design in order to alleviate the requirement for [H]-labeled reducing agents. In this work, we explore a suite of electrode or hydrogen gas driven catalyst systems for the generation of [4-H]-NADH and consider their use for driving reductive deuteration reactions.

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 H 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.

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

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

Carbon as a Simple Support for Redox Biocatalysis in Continuous Flow.

A continuous packed bed reactor for NADH-dependent biocatalysis using enzymes co-immobilized on a simple carbon support was optimized to 100% conversion in a residence time of 30 min. Conversion of pyruvate to lactate was achieved by co-immobilized lactate dehydrogenase and formate dehydrogenase, providing in situ cofactor recycling. Other metrics were also considered as optimization targets, such as low E factors between 2.

Biocatalytic hydrogenations on carbon supports.

We describe the use of carbon as a versatile support for H-driven redox biocatalysis for NADH-dependent CX bond reductions in batch and flow reactions. In each case, carbon is providing an electronic link between enzymes for H oxidation and reduction of the biological cofactor NAD as well as a support for a multi-enzyme biocatalysis system. Carbon nanopowders offer high surface areas for enzyme immobilization and good dispersion in aqueous solution for heterogeneous batch reactions.