Mass spectrometric determination of dioxygen bond splitting in the "peroxy" intermediate of cytochrome c oxidase

The "peroxy" intermediate (P form) of bovine cytochrome c oxidase was prepa red by reaction of the two-electron reduced mixed-valence CO complex with O -18(2) after photolytic removal of CO. The water present in the reaction mi xture was recovered and analyzed for O-18 enrichment by mass spectrometry, It was found that approximately one oxygen atom (O-18) per one equivalent o f the P form was present in the bulk water. The data show that the oxygen-o xygen dioxygen bond is already broken in the P intermediate and that one ox ygen atom can be readily released or exchanged with the oxygen of the solve nt water.