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

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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.