NextGen Structural Biology under Electrochemical Control: Filling in Missing Intermediates in Metalloenzyme Catalytic Cycles

Chemical reactions critical for a net-zero, renewable-energy future are the production and oxidation of hydrogen gas as a clean, renewable fuel, and the efficient production of ammonia for fertiliser or as a clean hydrogen storage system. Nature has already solved these chemical challenges, in the form of microbial hydrogenase and nitrogenase enzymes, which comprise clusters of earth-abundant metals wrapped up in a protein framework to enable use of hydrogen as a fuel or production of ammonia from nitrogen in the air. In this project we develop and apply a set of research tools, which allow us to fill in gaps in understanding of how these enzymes work, providing insight that will feed into wider research efforts to establish viable clean energy technologies to address the urgent climate challenge. We use x-rays and neutrons to collect a combination of static images (akin to 'photographs') and dynamic 'movies' of these enzymes as they carry out key catalytic steps, in order to understand how they achieve the splitting of strong chemical bonds in hydrogen and nitrogen. This will provide important information to assist biologists to understand the enzymes, and to assist chemists to design new catalysts for energy technologies.

X-rays are used routinely to provide images of the location of atoms in a complex enzyme molecule in the crystal state, where many molecules of the enzyme pack into an ordered array. Enzymes can perform their chemical reaction in the crystal and the last decade has seen exciting technical advances in synchrotron/laser x-ray sources and detectors that enable rapid collection of many x-ray 'images', offering possibilities of making 'movies' of how atoms move in enzymes as they function. However, such movies are only possible if all the enzymes in the crystal are held in the same initial state at the start of the reaction - equivalent to the challenge of aligning a team of unruly runners at the starting line before a race-and all react at the same time. This presents a second challenge, finding an appropriate trigger- equivalent to a starting gun used to begin a race - to start the reaction. Our previous work provides solutions to these challenges. Firstly, we have found how to use electrodes to apply an electrochemical potential to bring all the molecules into a uniform state - the same oxidation level- to start catalysis. Secondly, Ash has demonstrated light triggers can be applied to this uniform starting state to begin catalysis. During the project, we start by fine-tuning these control and trigger mechanisms, adapting them for the tiny crystals used in time-resolved x-ray methods. We then use electrochemical control to produce high quality static snapshots of each oxidation level of hydrogenase. We then apply the light triggers to initiate steps in catalysis, and record molecular movies of the enzyme in action. This will give the most detailed view ever achieved of hydrogenase actually working.

Next, we address a limitation in x-ray structural images that it is very difficult to pinpoint the location of the tiny hydrogen atoms which are released as the enzyme splits hydrogen gas. For this we turn to neutron beams to show up the elusive hydrogen atoms. Using very large crystals of hydrogenase, we again apply electrochemical control to trap the enzyme molecules at a uniform oxidation level, before firing neutrons at them to show the exact positions of the hydrogen atoms that are so critical in hydrogenase catalysis.

Finally, we turn to nitrogenase, showing that we can apply our electrochemical control and light triggers here too, demonstrating the broad applicability of our methods to different enzymes relevant to energy technologies. We aim to capture nitrogenase in action during binding, release or transformation of non-natural substrate molecules to better understand where and how nitrogen binds and is split.

This project establishes new tools for mechanistic study of redox metalloproteins by combining advanced structural and spectroscopic methods with electrochemistry, building upon the team's world-leading expertise in electrochemical control over redox state within hydrogenase single crystals. Serial xfel and synchrotron crystallography offer the possibility of both damage-free structures and time-resolved data collection to reveal details of atomic motions during metalloprotein reactivity. In order to build up 'molecular movies', methods for synchronously initiating chemistry in the crystalline state are needed. A notable omission from the current structural biologists' toolkit are ways of initiating rapid redox chemistry in the crystalline state. Here we address this gap, using electrochemical control to produce a uniform 'resting' state within crystals of NiFe hydrogenase, and initiating redox reactivity using rapid light triggers and 'caged' electron sources soaked into microcrystal samples. We will leverage known light-sensitive steps during hydrogenase catalysis to study proton-coupled electron transfer, using neutron diffraction and time-resolved IR spectroscopy to reveal details of protonation sites during hydrogenase catalysis. In combination with pH/pD-dependent measurements, this will allow us to interrogate the choreography of H+ and electron transfer in unprecedented detail, from both structural and spectroscopic perspectives.

By extending our tools to the complex nitrogenase enzyme, linking for the first time key CO-bound structures to spectroscopic data and revealing the first isocyanide-bound nitrogenase structures, we demonstrate wider applicability to other metalloproteins. This step-change in control of protein crystals will bring the UK to the forefront of bioinorganic serial synchrotron and xfel efforts, and our methods will interest bioinorganic and biophysical chemists, structural biologists and the wider xfel/crystallography communities.