Hydrogen evolution and oxidation reactions are common in the microbial world. They are catalysed at nickel-iron or di-iron catalytic sites within enzymes called hydrogenases, and allow microbes to vent excess reducing equivalents as H2, or to utilise H2 as a food/fuel source. Hydrogenases are being applied in biocatalytic heterogeneous hydrogenations (ChemCatChem 2015, 7, 3480) and have been demonstrated in H2/O2 enzyme fuel cells (eg Energy Environ Sci 2013, 6, 2166). Studies to elucidate their mechanism also promise fundamental insight into how to activate or form H2 at cheap, non-noble metal catalyst sites and thus pave the way for cheaper, bio-inspired synthetic catalysts for energy chemistry. Fundamental studies into the hydrogenases carried out on this project therefore underpin technologies to establish H2 as a renewable fuel for the future. This project falls within the EPSRC Energy research area, in particular addressing the Alternate fuels and Renewable energy sub-themes. This project applies new infrared spectroscopic methods to study the mechanism of NiFe hydrogenase enzymes. We aim to pinpoint the sites involved in proton transfer from the active site during H2 oxidation (specific amino acids or ordered watered molecules), and understand the coupling of proton and electron transfers. We aim to understand differences between hydrogenases from different micro-organisms or with varied cellular roles in order to define the core mechanistic steps as well as variations in the mechanism. These studies will inform a new generation of bioinspired hydrogen-energy catalysts that build in proton-transfer relays around a metallo-site. Research on this project will exploit sophisticated ways of coupling IR spectroscopy with in situ electrochemical control developed in the Vincent group. First we exploit an approach termed Protein Film Infrared Electrochemistry (PFIRE), in which the hydrogenase is immobilised on an electrode surface and IR spectra are recorded during electrocatalytic turnover of the enzyme. This reports on the steady state distribution of active site states present during catalysis (Angew Chemie Int Ed 2015, 54, 7110), and is proving powerful in addressing the chemistry of metalloenzymes and in providing mechanistic insight (J Phys Chem B 2015, 119, 13807). To access more transient intermediates in the catalytic cycle, the project will continue to develop a recently demonstrated IR microspectroscopy method that exploits electrochemical control over single crystals of hydrogenase. Slowed chemical steps in the crystalline state permit spectroscopic observations of intermediates that are otherwise interconverted too rapidly to detect in solution (Chem Commun, 2017, 53, 5858). As well as experiments conducted in the Vincent group's well-established IR labs, the project will make use of IR microspectroscopy beamline MIRIAM at Diamond Light Source.