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 UVpump-IRprobe spectroscopy to study the reversible photochemical activation of the CO-inhibited Hox-CO state over picosecond to millisecond time scales. Since this process transforms the catalytic site into the active, H2-binding Hox state, photolysis of Hox-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 Hox-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.
Kobeleva et al. (2026) studied this question.