Anaerobic time-resolved serial crystallography captures CO dissociation and rebinding in oxygen-sensitive [FeFe]-hydrogenase

Redox-active, oxygen-sensitive metalloenzymes catalyze key reactions in biological energy conversion and small-molecule activation. Understanding their mechanisms requires structural characterization of transient catalytic intermediates. Time-resolved serial crystallography (TR-SX) enables direct visualization of protein dynamics during catalysis under near-physiological conditions. Its application to oxygen-sensitive enzymes has remained challenging because strict anaerobic conditions must be maintained throughout sample preparation and data collection. Here, we establish an anaerobic room-temperature serial crystallography workflow for the oxygen-sensitive [FeFe]-hydrogenase Cp I and demonstrate its applicability by determining a room-temperature structure of the CO-inhibited H _ox -CO state and following inhibitory CO dissociation and rebinding on the millisecond timescale via TR-SX. The presented room-temperature H _ox -CO structure closely resembles previous cryogenic models and shows no detectable evidence of oxygen-induced degradation or significant radiation damage. Time-resolved measurements reveal no detectable structural rearrangements accompanying CO dissociation and rebinding beyond displacement and return of the inhibitory ligand, indicating a rigid catalytic architecture that may facilitate rapid catalysis. The presented workflow enables time-resolved structural studies of oxygen-sensitive metalloenzymes under physiologically relevant conditions and opens the way to direct visualization of catalytic intermediates in redox enzymes.