Detection of a Geminate Photoproduct of Bovine Cytochrome Oxidase by Time-Resolved Serial Femtosecond Crystallography.

Cytochrome oxidase (CO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme group in their active sites, CO has a unique binuclear center (BNC) composed of a copper atom (Cu) and a heme iron, where O binds and is reduced to water. CO is a versatile O surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CO (bCO) revealed that photolyzing CO from the heme iron leads to a metastable intermediate (Cu-CO), where CO is bound to Cu, before it escapes out of the BNC. Here, with a pump-probe based time-resolved serial femtosecond X-ray crystallography, we detected a geminate photoproduct of the bCO-CO complex, where CO is dissociated from the heme iron and moved to a temporary binding site midway between the Cu and the heme iron, while the locations of the two metal centers and the conformation of Helix-X, housing the proximal histidine ligand of the heme iron, remain in the CO complex state. This new structure, combined with other reported structures of bCO, allows for a clearer definition of the ligand dissociation trajectory as well as the associated protein dynamics.