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

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

Renewable hydrogen offers us a future free of fossil fuels, not just because we might use hydrogen in our cars, but because hydrogen is the primary chemical formed upon water energisation by sunlight and is used to make other energy-rich chemicals. Hydrogen, the simplest of molecules is also one of the most important industrial chemicals and more than 70 million tonnes are used worldwide. It is a raw material for some of the most important chemical processes, particularly in making ammonia, the essential fertiliser. At present, most hydrogen is produced from fossil fuels; however, hydrogen is also the greenest and most easily renewable of future fuels and raw materials because sunlight and water are earth-abundant resources. We are familiar with the electrolysis experiment in the school laboratory where hydrogen is formed along with oxygen because electrical energy is converted to chemical energy: explosive recombination of the hydrogen and oxygen, initiated by a spark, releases back much of the original electrical energy as heat and light. Solar energy also, can be stored as hydrogen; indeed, green plants do this in a disguised way in photosynthesis (consider that 'hydrogen' is 'stored' by combining it with carbon dioxide to give hydrocarbons and carbohydrates). Energy from the sun is easily able to convert water from the oceans into hydrogen and oxygen yet we this does not happen at any detectable rate: converting water to hydrogen requires not only systems for absorbing radiative energy (pigments, semiconductors) but also catalysts that will accelerate the chemical reactions.
This research project is about the catalysts, produced by microorganisms, that convert water into hydrogen, and vice versa, at rates of many thousands per second at normal temperatures. These catalysts are giant molecules - enzymes known as hydrogenases - and they are of great importance for understanding and designing the chemistry of future hydrogen technologies. It is through hydrogenases that microbes thrive in all kinds of different environments and produce hydrogen (biohydrogen) for human benefit. Conversely, hydrogenases are important for the action of some notorious pathogens.
Use of hydrogen in current industrial applications requires high temperatures and expensive resources. In terms of performance, the best catalysts available to industry are based on platinum, a limited, expensive element. In contrast, hydrogenases catalyse the interconversion between hydrogen gas and protons (water) at rates and efficiencies higher than platinum but using the common elements iron and nickel. To achieve these rates, nickel and iron are 'dressed up' in special atomic environments that are also buried to shield them from water and other small molecules that may disrupt or destroy the special environment. One of the specific aims of this research is to establish how the special structures of the active sites of the two kinds of hydrogenase (one contains only iron, the other contains iron and nickel) perfected by biology during over two billion years of evolution, lead to such high activity. How does hydrogen interact with the atoms of the active site, how important is the exact positioning of different atoms, how important are bond strengths and mobilities of different groups, what special properties of other small molecules enable them to block the normal reactions with hydrogen ? With this information we can (a) determine definitive rules for the design of synthetic catalysts, based on iron or nickel having activities as high as platinum, (b) engineer hydrogenases so that they can survive oxygen, leading eventually to sustainable, large scale photosynthetic hydrogen production by whole organisms, (c) engineer hydrogenases so that they can be applied, as isolated enzymes, in special technologies such as fuel cells and in continuous 'cofactor regeneration' a technical requirement for enzyme-based synthesis of some expensive chemicals.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50

publication icon
Armstrong FA (2018) Protein Film Electrochemistry of Iron-Sulfur Enzymes. in Methods in enzymology

publication icon
Bachmeier A (2015) Solar-driven proton and carbon dioxide reduction to fuels-lessons from metalloenzymes. in Current opinion in chemical biology

publication icon
Bachmeier A (2014) Selective visible-light-driven CO2 reduction on a p-type dye-sensitized NiO photocathode. in Journal of the American Chemical Society

 
Description We discovered a key role for an arginine residue in the active site. The arginine has a long sidechain that can block the site to ligands apart from hydrogen. We proposed that the guanidine group may function as the catalytic base in hydrogen oxidation. We consolidated a model for catalytic bias in hydrogenases and discovered how to convert a hydrogen-oxidising hydrogenase into an effective hydrogen producer. We established how two intermediates of hydrogen oxidation by [NiFe]-hydrogenases are in a pH-dependent equilibrium. We have since gained more evidence for an important role of the pendant arginine as well as the importance of residues in the 'canopy' region, as well as consolidating the catalytic bias model. These results have fundamental importance for understanding how to activate the simplest of all molecules that is also the focus for future solar energy capture via catalytic water splitting.
Exploitation Route The possible role of a guanidine group as the catalytic base in [NiFe]-hydrogenases has started to attract a good deal of attention.
Sectors Chemicals,Energy

 
Description How Hydrogenases Work at the Atomic Level
Amount £722,942 (GBP)
Funding ID BB/N006321/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2016 
End 03/2019
 
Description Invited Lecture - OXF 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited lecture at GRC on Metal Ions in Biology, Ventura, California, January 2017
Year(s) Of Engagement Activity 2017
 
Description Invited Lecture - OXF 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Sparked discussion

Ideas about collaborative funding
Year(s) Of Engagement Activity 2014
 
Description Invited lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited lecture at the Gerischer-Kolb Symposium, Reisenberg Castle, Germany, October 11-13, 2017
Year(s) Of Engagement Activity 2017
 
Description Invited lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Lecture at Workshop on Energy Materials Research, Berlin 10-11 October 2016
Year(s) Of Engagement Activity 2016
 
Description Invited lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited lecture at the Ernst-Haage Symposium on Chemical Energy Conversion, Mulheim, Germany, Noverember 22-24, 2016
Year(s) Of Engagement Activity 2016
 
Description Invited lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited lecture at The 7th Life Science Symposium 'Bioenergy', Delft, Netherlands, May 10, 2016
Year(s) Of Engagement Activity 2016
 
Description Invited lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Lecture at DehaloCon II, Leizig Germany, 2017, March 26 to 29
Year(s) Of Engagement Activity 2017
 
Description Swift Lecturship 2014 at Caltech - OXF 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This was a prestigious lecture presented to leading eminent researchers. The title was 'The Chemistry of Biohydrogen'

Scientific discussions, throughout the week I was at caltech
Year(s) Of Engagement Activity 2014