Abstact

Iron-sulfur (Fe-S) clusters are ubiquitous as redox-active protein cofactors, but it is often difficult to collect protein structures in which redox centres are in uniform and well-defined oxidation states. Using spinach ferredoxin I (Fdx) as a model redox protein, we demonstrate an integrated methodological pathway for electrochemical modulation of redox state in protein crystals coupled with in crystallo EPR and online-UV-visible spectroscopy to verify oxidation state. We show that Fdx crystals can be electrochemically reduced, reversibly, without compromising lattice integrity or X-ray diffraction quality. We show that redox levels can be precisely ascertained in crystallo via EPR and UV-visible spectroscopy, enabling a direct correlation between protein structure and electronic state of the metal cluster. In this way, we generate and compare 'oxidised', 'reduced' and 're-oxidised' structures of Fdx. Overall, our approach demonstrates a pipeline which will be applicable to structure-function studies of a wide range of electron-transfer proteins and redox enzymes.