How hydrogenases work at the atomic level

Renewable energy is synonymous with exploiting sunlight, but the sun does not always shine. In green plants, energy from sunlight is stored by using it to convert water and carbon dioxide into sugars and oxygen. Scientists are very interested in discovering ways of mimicking the processes occurring in the green leaf, not so much to produce sugars but to make hydrogen (H2) by splitting water an abundant resource. Hydrogen, the smallest of all molecules, is therefore the most important primary fuel of the future: aside from using sunlight itself, H2 can be produced from any common source of electrical energy (such as surplus electricity from windfarms). The energy stored in H2 is released either by combustion or by its conversion back into electricity in fuel cells, but H2 is also a raw material for making fertilisers and other chemicals including liquid fuels for transport.
In learning how to use and produce H2 most efficiently, at low cost, scientists are looking again to nature itself. Much progress has recently been made in discovering how effective microorganisms are in producing and using hydrogen as a metabolite, and many details of the mechanism by which enzymes known as hydrogenases catalyse this reaction are now known. 'Activating' hydrogen means making or breaking the chemical bond between two hydrogen atoms. Electrons are moved into or from the hydrogen molecule from other atoms in the active site of hydrogenases, and just tiny distances are critical. The enzymes have evolved to have every atom of the active site in just the right place.
The research to be carried out will identify the positions and roles of each of the different atoms of the active site of hydrogenases, leading scientists to a 'blueprint' by which to create the hydrogen catalysts of the future. To determine this information at the atomic level requires genetic engineering to be combined with structural definition at the highest resolution possible using x-ray diffraction. It is not easy to detect the positions of hydrogen atoms by x-rays and this is particularly the case when they are in giant molecules like enzymes: therefore an important challenge will be to achieve extremely high resolution by careful attention to sample homogeneity and crystallisation. Efforts will also be made to test the feasibility of obtaining a structure of a hydrogenase 'trapped in action' by making a genetically-engineered variant in which H2 is bound but cannot react further. The ultimate goal of the research would be to see if the structure of such a 'trapped in action' hydrogenase can be determined using neutron diffraction, as neutrons are able to pinpoint the accurate position of hydrogen atoms, making it possible to observe the hydrogen molecule itself.

The research will determine, at atomic resolution, the mechanism by which a class of enzymes known as [NiFe]-hydrogenases split the bond in the hydrogen molecule to release two electrons and two protons. This reaction, managed so well by biological catalysts, is of intense interest in the development of renewable energy that is based on using sunlight or surplus electricity to split water, producing H2 and O2. The H2 can be used directly to give back energy or as a raw material for the chemical industry and for producing liquid fuels by reaction with CO/CO2.
The [NiFe]-hydrogenase known as Hyd-1 and produced by E. coli has proved to be an incredible model for understanding the mechanism of enzymatic hydrogen activity. We have now categorised most of the intermediate states in the catalytic cycle and most recently, in research carried out in Oxford, we have established the importance of amino-acids, previously ignored yet lying in the active site, that are actually responsible for breaking/making the H-H bond. We are now reaching the stage in which atomic-level detail, necessary for transferring this knowledge into the most efficient synthetic catalysts of the future, is starting to emerge, for different stages of the catalytic cycle. The research will combine the advances being made through genetic engineering and kinetic characterisation of Hyd-1 variants at Oxford with high-resolution structure determination of these variants at different states of the catalytic cycle, using Diamond Light Source. An ultimate aim is to pinpoint the binding of molecular H2 in the 'Michaelis complex' formed with a variant of Hyd-1 unable to progress beyond this stage of the catalytic cycle. Such a structure is possible at the highest resolution available to X-rays, but we aim to investigate the feasibility of using neutron diffraction, for which an intermediate of hydrogenase represents the ultimate enzyme challenge.

PROJECTED IMPACT BASED ON THE AIMS & OBJECTIVES OF THE PROJECT
As well as resolving a fundamental chemistry question, establishing how hydrogen can be made and used efficiently given the perfect chemical environment, the research will ultimately have important and possibly revolutionary economic impacts in the energy sector leading to much improved production of renewable H2. It is expected that the outcome could also play a role in influencing government energy policy by providing greater confidence in our ability to develop hydrogen based on sounder scientific principles. The project has the following scientific aims:
1. Atomic resolution of the mechanism by which the H-H bond is formed or cleaved in [NiFe] hydrogenases, helping to direct the design of new catalysts. 2. To provide a model use of arginine as a special catalytic base in enzymes. 3. Determination of the mechanism of proton-coupled electron transfer, through high-resolution, in vacuo X-ray diffraction - the migration of a metal-bound hydride as a proton. 4. To establish the feasibility of using neutron diffraction to study biological hydrogen activation.


Application and Exploitation
Oxford University has a wealth of experience in engaging with industry. The Oxford Technology Transfer Office ISIS Innovation (http://www.isis-innovation.com/) deals with commercial exploitation through IP patenting, spin-out, and licensing. All exciting discoveries will be protected before being presented at conferences or published.

Communications and Engagement
The main data and conclusions of this work will be published in high-quality journals. The most exciting discoveries, ideas, and inventions will be protected. The public will be engaged at many different levels. Oxford Chemistry and Biochemistry have dedicated media offices, and we will communicate all exciting findings and publications through these channels. 'Outreach and Impact' pages are on websites that are open to the public to view e.g. http://outreach.chem.ox.ac.uk/
Oxford has a long tradition of public engagement. The popular Café Scientifique (http://www.cafesci.org/oxford/) events regularly bring academics together with the public in a relaxed café-style atmosphere to discuss their research. As an example of recent success, the Armstrong group was selected to exhibit at the prestigious Royal Society Summer Science Exhibition during July 1-7, 2013, with a stand entitled 'Solving the Energy Crisis - From Ancient to Future Solar Fuels'. We will contribute to the Oxford Science Open Days, which include practical demonstrations to the general public.

Collaboration
The project brings together experts in molecular biology, electrochemistry, and spectroscopy, who are dedicated to understanding the mechanism of hydrogenase, AND experts in enzyme crystallography who use Diamond Light Source (DLS) and see important possibilities for using neutron diffraction to study an enzyme that deals with molecular and atomic/ionic hydrogen. The researchers and their facilities (molecular biology instrumentation and services, electrochemistry, pulse EPR spectroscopy, IR spectroscopy, synchrotron diffraction at Diamond Light Source/Research Centre at Harwell) are located close together, making this team unique worldwide in having every technique available to solve the hydrogenase mechanism at the most fundamental levels.

Capacity and Involvement
Impact activities will be carried out at all possible levels. All Research Council-funded postdocs and DPhil students at Oxford are aware of the importance of public engagement and of maximising the impact of government-funded research. The Oxford technology and press offices will be used to support technological exploitation and public engagement. Career development of researchers is a key aim of this project and personal development programs are in place (http://www.skillsportal.ox.ac.uk/).