We have used EPR and FTIR spectroscopy in combination with 1 7 O and 1 5 N stable isotopes to investigate the mechanism of cytochrome c oxidase (CcO). A high-spin state of heme a 3 was found in high yield by EPR, achieved upon turning over the enzyme until it was anaerobic, and shown to be a mixture of heme with a coordinated oxygen-based ligand and five-coordinate heme. Allowing the enzyme to consume 1 7 O 2 for a few milliseconds before freezing, we also showed that the product H 2 1 7 O exits toward the external side of the enzyme, binding to the nonredox active Mg/Mn site en route.Specific 1 5 N labeling of histidine, in comparison with global 1 5 N labeling and unlabeled samples, allowed us to more definitively assign heme and histidine peaks in the electrochemically induced FTIR difference spectrum. Additionally, the assignment of heme bands affords a reliable method of spectrum normalization between samples, providing a more accurate comparison of the spectral features of bovine with bacterial cytochrome oxidase and revealing multiple differences between the two species.