Metal-hydrido intermediates in enzymes: atomic level mechanistic insight and technological applications of hydrogenases

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Hydrogenases are enzymes that oxidise or produce hydrogen (H2) at extremely high rates. They are under intense investigation because they provide detailed mechanistic insight for future catalyst development, they are paradigms for novel metal-hydrido intermediates in enzymes, they are crucial for renewable biological H2 production, and they can be incorporated into novel technologies. This research will consolidate new discoveries on the mechanisms by which hydrogenases interact with H2 and other small molecules at both the active site- and macromolecule level, and will develop methods for engineering hydrogenases for special applications. The research brings together scientists with expertise in many different fields - enzymes, molecular biology, spectroscopy, electrochemistry and computational chemistry. The research also offers expert training for young scientists destined for scientific careers. The enzymes are the (normally) membrane-bound [NiFe]-hydrogenases Hyd-1 and Hyd-2 from E.coli, and soluble [FeFe]-hydrogenases from various organisms. All are well-behaved from the viewpoints of molecular biology and enzyme structure, allowing design and production of specific variants to answer key scientific questions or apply in new technologies. Underpinning the physical measurements is protein film electrochemistry (PFE) which has revolutionised the study of hydrogenases by providing essential insight at two crucial stages of investigation, 'wide-angle' reconnaissance and 'focused' measurements of kinetics/energetics. Both EPR and FTIR will be used to characterise important active site states pinpointed by PFE. Computational chemistry will be used to help understand the results. Four subprograms will deal with: determination of the mechanism of H2 activation by [NiFe]hydrogenases; the oxidation of H2 by E.coli; unravelling two important new discoveries made with [FeFe]-hydrogenases; and engineering Hyd-1 and Hyd-2 for special technology applications.

This research will benefit a wide range of scientists and technologists and influence policy makers. Hydrogen is such an important molecule, in coordination and theoretical chemistry, fuel science, renewable energy, enzymology and microbiology, that many discoveries we will make will have some use, either in principle or in practice. The techniques being used will also be advanced by this work which provides some superb opportunities for their demonstration and exploitation. Protein film electrochemistry is going from strength to strength as advanced models are developed for the detailed interpretation of data; pulse EPR methods are solving challenging problems about the nature of paramagnetic intermediates and trapped states of enzyme active sites; the FT-IR spectroscopy currently being developed by Dr Vincent has state-of-the-art sensitivity and potential resolution; computational chemistry will be applied to the mechanism of activation of the smallest molecule. As with our recent research, which (a) played a major role in identifying and characterising a new type of iron-sulfur cluster (generating a Fe-N bond during two-electron redox cycling), (b) resulted in the discovery of a novel, reversible inhibition of hydrogenase by formaldehyde, and (c) proved that an O2-tolerant [NiFe]-hydrogenase is a hydrogen oxidase, we expect to make further discoveries that will have a lasting influence on science. Hydrogenases are the paradigms for metal-hydrido intermediates in enzymes, a more reactive hydride transfer agent than flavins or NAD(P)H, yet until now little known, let alone understood. It is possible that Fe-H and Ni-H species are the active intermediates in nitrogenase or carbon monoxide dehydrogenase, and our research will be directly relevant to these other pressing mechanistic challenges.
Discoveries resulting from our research on the in vivo properties and reactions of hydrogenases will be valuable for microbiologists and those working on pathogens where oxygen tolerant hydrogenases are important. The possibility of a whole cell hydrogen sensor is also of potential commercial interest.
If we are successful in determining how to improve the oxygen tolerance of [FeFe]-hydrogenases, then the outcome could be to ignite much more interest in the possibility of photosynthetic hydrogen farms. If we succeed in structurally defining the nature of the aldehyde adduct formed with [FeFe]-hydrogenases, we will have a powerful new probe not only of active site chemistry but also (with long-chain analogues) the tunnels through which small molecules travel to the active site. The possibility that hydrogenases could be engineered to catalyse the hydrogenation of small molecules is an exciting possibility that may have commercial applications.
One of the enzymes being studied in terms of its coupling to materials, the oxygen tolerant 'Hyd-1' is already earmarked for technological development. It is a stable H2 oxidiser that can be genetically modified. We have already identified its use in novel fuel cells with bilirubin oxidase as O2 reducer. Hyd-1 is now likely to be a fine candidate for the continuous hydrogen-driven NADH cofactor regeneration being developed by Kylie Vincent with the aim of commercialisation. Another enzyme, Hyd-2, is a good H2 producer and if we can modify its surface for stable attachment to semiconducting nanoparticles we will have a superb model for solar hydrogen production studies, of interest to those working in renewable energy-artificial photosynthesis.
The more we learn about hydrogen - how it can be produced easily from sunlight or electricity using abundant elements as catalysts, the convenience of conducting ambient-temperature transformations, its application to novel technologies and its role in microbial life - the more likely it is that policy makers and industry itself will take hydrogen seriously. Success in this direction alone would be a very important impact outcome.