How hydrogenases work at the atomic level

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

Abstract

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.

Technical Summary

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.

Planned Impact

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/).

Publications

10 25 50

publication icon
Wulff P (2016) How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure. in Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry

publication icon
Evans RM (2016) Mechanism of hydrogen activation by [NiFe] hydrogenases. in Nature chemical biology

publication icon
Carr SB (2016) Hydrogen activation by [NiFe]-hydrogenases. in Biochemical Society transactions

publication icon
Pandey K (2017) Frequency and potential dependence of reversible electrocatalytic hydrogen interconversion by [FeFe]-hydrogenases. in Proceedings of the National Academy of Sciences of the United States of America

 
Description We have examined the influence of each of the active site 'canopy' residues on the catalytic properties of an oxygen-tolerant [NiFe]-hydrogenase called Hyd-1 from E.coli. We have also successfully started parallel investigations on the oxygen sensitive counterpart known as Hyd-2. We have replaced the active site arginine in Hyd-2 by lysine, producing a variant (R470K) that is equivalent to that produced (R509K) in Hyd-1. In both cases, removal of the arginine, which places a guanidinium group less than 4.5 A from the Ni, lowers the activity by 100-fold, while also greatly lowering the activation energy: the interpretation is that the energy barrier is replaced by an entropic barrier, meaning that an essential group is not in the optimal position for catalysis. The results support our proposal that the guanidinium group plays an key part in catalysis, and this role is likely to be as the catalytic base, in what is known as a Frustrated Lewis Pair (FLP) mechanism. Important crystal structures have been determined at Diamond Light Source. The structure of Hyd-2 R479K reveals a diatomic ligand bound to the Ni atom, in enzyme samples expose to air. The diatomic molecule looks to be O2, which if true represents a major discovery; we are now carrying our Raman experiments to verify if we have an Nickel-O2 analogy to the classic case of O2 bound to hemoglobin. We have investigated the role of a residue E28, which has long been known to be important in transferring protons in and out of the active site. In Hyd-1 we have mutated the glutamate to a glutamine: the E28Q variant is virtually inactive at pH 6, but the pH stability of this enzyme has allowed us to study its activity up to pH 11, where the enzyme is quite active. Even at pH 6, E28Q becomes active when an oxidising potential is applied, and we can correlate this with the superoxidation of the unusual proximal [4Fe-3S] cluster. The crystal structure shows that when this cluster is in the superoxidised state, a water molecule appears close to the glutamine headgroup. We thus have a unique study showing ways in which a proton barrier in an enzyme can be breached. Detailed analysis of the steady-state IR spectra using a technique called PFIRE has shown that the initial activation (H-H cleavage) reaction in the E28Q variant is fast, but reoxidation of reduced intermediates is slow: this proves that E28 (which lies close to the Ni-thiolate site) is not involved in the initial step in which the H-H bond is cleaved, but in a later stage in which the 'hydridic' H atom is removed. We have now produced numerous crystal structures of Hyd-1 and Hyd-2 and their active site variants. We have succeeded in detecting catalytic H-atoms attached to Ni, or to Ni and Fe, but we have not detected any H-atoms attached to a cysteine ligand, as was claimed in a recent paper by a Ogata et al Nature 520, 571-574 (2015). Therefore, all our evidence so far points against H2 activation using a thiolate ligand as the direct base. In recent work that has just been accepted for publication in PNAS, we have mutated all four Ni-coordinating cysteines to selenocysteine. We have identified one position as being particularly important for O2 tolerance. In investigations of the equivalent arginine mutation in Hyd-2 we have identified a tightly bound diatomic ligand. Final investigations are underway to establish whether or not this ligand is an O2 molecule.
Exploitation Route Anyone working on biological hydrogen should benefit from our research
Sectors Chemicals,Energy,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
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 
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 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 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 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