Single protein crystal spectroscopy and crystallography of hydrogenase under electrochemical control

Proteins are the nanoscale 'machines' that control almost all processes in cells. Importantly they are responsible for speeding up (catalysing) chemical reactions that make the essential molecules for life and release or store energy. Understanding the structures of individual proteins at the atomic level has been absolutely key in building up our understanding of how they contribute to the function of cells. In particular, the technique of X-ray crystallography has been extremely valuable in providing 'snapshot' images of many different proteins at the level of individual atoms. In this approach, crystals of the protein are prepared, and are probed using a high energy X-ray source which diffracts off
individual atoms in the crystal, giving a pattern that reveals the positions of atoms in the protein. A series of these 'snapshots' is needed to understand each step in how a protein works, and it is often difficult to trap proteins in specific states relevant to their function in order to obtain a complete set of 'snapshots'. This is particularly true for a group of 'redox' proteins which catalyse chemical reactions involving the transfer of electrons - oxidation and reduction reactions. A further challenge arises because these redox proteins often incorporate metal atoms that are susceptible to damage by X-rays during the data collection for crystal structure determination. It is very timely that we find new tools for studying these sort of proteins, because they catalyse many chemical reactions which are relevant to solving big global challenges, including how to use hydrogen as a sustainable fuel, how to capture the greenhouse gas carbon dioxide and turn it into useful chemicals, and how to efficiently produce the fertilisers needed to sustain the world's growing population.

In this project, we demonstrate a completely new approach to controlling and verifying the state of redox proteins that will allow crystal structure snapshots to be produced for many more of the important functional states of these redox proteins. Our focus is a protein called hydrogenase which allows microbes to live on hydrogen gas as their energy source. In earlier preliminary work, we have shown that we can use electrodes to control a single crystal of hydrogenase to generate uniform states relevant to its function. This provides an unprecedented way to get proteins in single crystals into specific states ready to record X-ray crystallographic 'snapshots'. At the same time, we make use of imaging using infrared light with a special infrared microscope to confirm the state of the protein in the crystal. During the project, we will show that we can prepare specific states of protein crystals in this way and then record their X-ray structures to yield snapshots of previously unseen states of the protein. This will yield new information on how proteins function as efficient catalysts for the important reactions mentioned above. We will also use the infrared imaging approach to check the crystals after exposure to X-rays to make sure that the state of the protein in the crystal has not been damaged during X-ray crystallographic data collection. This approach will lead to much more reliable snapshots of redox proteins.

The project thus represents a step change for structural biology of redox proteins and understanding the function of proteins which may teach us how to solve important global problems. We would like these tools to become widely available to structural biologists who solve the structures of complicated proteins, and during this project we aim to develop our approaches so that they can be readily implemented.

What we learn about the way that hydrogenases work in the course of this project will help to propel biotechnological applications of hydrogenases, and will underpin development of alternative chemical catalysts based on the cheap metals, nickel and iron, found inside the hydrogenases.

The project establishes new tools for metalloprotein structural and mechanistic studies by combining infrared (IR) microspectroscopic imaging with electrochemical control over the redox state of protein in a single crystal. We address two key problems in X-ray crystallography of metalloproteins: the difficulty in generating and verifying well defined states of redox protein crystals, and concerns over X-ray damage during data collection. Focussing on E. coli NiFe hydrogenases, in which CO and CN- ligands at the bimetallic active site give rise to strong absorption bands in the IR that are very sensitive to the redox state of the active site, we demonstrate that it is possible to control single protein crystals in an electrochemical cell, and simultaneously to image, using IR microspectroscopy, the redox states generated at each potential. This enables us to manipulate single protein crystals, electrochemically, into specific redox levels and then trap by freezing. IR imaging before and after collection of electron density maps will be used to determine whether X-ray exposure has changed the state of the protein. Furthermore, we take advantage of slowed proton transfer and reaction dynamics inside a protein crystal to resolve steps in metalloenzyme reactivity that are too fast to resolve in solution using conventional stopped flow or freeze quench approaches, trapping out specific proton-transfer states of the hydrogenases by freezing crystals for EPR, IR and X-ray crystallographic study. This will be particularly valuable where certain catalytic intermediates exist only for microseconds in solution, but may be spectroscopically accessible in crystalline samples. We exploit this suite of approaches to understand the successive movements of protons at the NiFe active site during hydrogenase catalysis. Overall, the tools developed will have wide applicability in structural biology and bioinorganic chemistry.

The project has substantial and immediate academic impact for the structural biology and bioinorganic chemistry communities. We develop new tools for controlling and imaging metalloprotein single crystals with unique benefits for structure-function and reactivity studies.

The electrochemical control strategy we establish for single metalloprotein crystals makes it possible to precisely manipulate crystals into well-defined redox levels for structural and spectroscopic study. The integrated IR imaging is highly sensitive to coordination at metal centres in proteins and to oxidation state and does not damage crystals. With IR imaging it is also possible to verify whether the redox state of the protein across the crystal has been altered during collection of X-ray data. Together, these developments address two key concerns in metalloprotein crystallography: how to achieve X-ray structures in well-defined redox states, and how to prove that samples have not suffered X-ray damage during data collection.

We also integrate electrochemical control over single crystals with EPR spectroscopy, enabling examination of electronic structure for well-defined redox states. The ability to conduct IR, EPR and X-ray crystallography on the same protein crystal provides unique opportunities to unify insight from different structural/spectroscopic approaches.

To ensure effective knowledge exchange and awareness of these new tools, we plan an intensive program of dissemination, including EuroBIC, ICBIC, RSC Dalton Meeting, Inorganic Biochemistry Discussion Groups, crystallography, biophysics and synchrotron meetings. As well as high-impact, open access publications, we will prepare 3 video podcast demonstrations of the methods for promotion on Univ. Oxford website and YouTube channel (90k followers), aimed at academics, public and students. We also intend to work with a commercial supplier to make our electrochemical-IR imaging cell available so it can be implemented readily by structural biologists, and Impact Acceleration funds will be sought to support this. We will provide a custom-built electrochem./micro-imaging cell to beamline B22 at Diamond Light Source for general use to benefit UK structural biologists.

Reactivity studies that exploit slowed proton transfer and reaction dynamics in protein crystals to enable imaging of transient intermediates in metalloprotein reactions will have further academic impact in the bio-inorganic chemistry community by enabling new levels of understanding of metalloprotein catalysis or sensing.

Understanding of proton transfer in NiFe hydrogenases arising from the project will propel applications of hydrogenases (eg in a H2-driven biocatalysis technology which we are separately translating to market) and inform the development of synthetic catalysts which incorporate bio-inspired proton relays. The techniques developed are applicable much more widely, and are particularly valuable for metalloenzymes that catalyse small molecule activation, many of which are relevant to energy chemistry (including sustainable fuels, H2 chemical storage, chemicals from CO2). The project should thus have longer term (5-10 year) environmental benefits: propelling research into new generations of catalysts for addressing key energy challenges. We will ensure knowledge transfer to this sector by presenting at chemistry/catalysis conferences, preparing broad-perspective review articles and engaging with Oxford Energy Network. For the researcher Co-Is, the project provides excellent cross-disciplinary training, exposure across several academic communities, and experience in collaborating across field boundaries. Immediate societal benefits will come from improved public science understanding through schools and public outreach on structural biology and imaging. Longer term societal benefits will arise from new tools for understanding of metalloprotein function with impacts across healthcare, biotechnology and bioenergy.