Photoactivation of [FeFe] Hydrogenase Studied by Multiscale Time-Resolved Infrared Spectroscopy

[FeFe] hydrogenases catalyze the reversible cleavage of H₂, a clean fuel, at exceptional rates. Therefore, understanding their catalytic mechanism is of high importance. Here, we employ multiscale UV_pump-IR_probe spectroscopy to study the reversible photochemical activation of the CO-inhibited H_ox-CO state over picosecond to millisecond time scales. Since this process transforms the catalytic site into the active, H₂-binding H_ox state, photolysis of H_ox-CO represents a unique strategy for studying light-triggered activation and the catalytic cycle of the enzyme with high time resolution. We show that the extrinsic CO of H_ox-CO dissociates in picoseconds and remains unbound for up to milliseconds. During this time, the enzyme is available for H₂ binding and further catalytic transformations. This time window is sufficiently large to study catalytic processes from the earliest steps to completion of the catalytic cycle. Our approach provides a basis for investigating the catalytic cycle of [FeFe] hydrogenases in real time and without diffusion limitation.