The use of stable isotopes and spectroscopy to investigate the energy transducing function of cytochrome c oxidase

We have used EPR and FTIR spectroscopy in combination with (17)O and (15)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 (17)O(2) for a few milliseconds before freezing, we also showed that the product H(2)(17)O exits toward the external side of the enzyme, binding to the nonredox active Mg/Mn site en route. Specific (15)N labeling of histidine, in comparison with global (15)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.