The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase
Philip A. Ash, Sophie E. T. Kendall-Price, Rhiannon M. Evans, S.B. Carr, Amelia Brasnett, Simone Morra, Jack S. Rowbotham, Ricardo Hidalgo, Adam J. Healy, Gianfelice Cinque, Mark D. Frogley, Fräser A. Armstrong, Kylie A. Vincent
Abstract
ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle.