Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography

Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, CcO has a unique binuclear center (BNC) comprised of a copper atom (Cu(B)) and a heme a(3) iron, where O(2) binds and is reduced to water. CO is a versatile O(2) surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a(3) iron leads to a metastable intermediate (Cu(B)-CO), where CO is bound to Cu(B), before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the heme a(3) iron and moved to a temporary binding site midway between the Cu(B) and the heme a(3) iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a(3) iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.