title: The use of stable isotopes and spectroscopy to investigate the energy transducing function of cytochrome c oxidase.
abstract: 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.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing MI 48824, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Schmidt
authors.forename: Warwick
authors.initials: W
authors.lastname: Hillier
authors.forename: John
authors.initials: J
authors.lastname: McCracken
authors.forename: Shelagh
authors.initials: S
authors.lastname: Ferguson-Miller
mesh_terms: D000818:Animals; D002417:Cattle; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D004734:Energy Metabolism; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008954:Models, Biological; D009587:Nitrogen Isotopes; D010103:Oxygen Isotopes; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D009587:Nitrogen Isotopes; D010103:Oxygen Isotopes; C027728:heme a; D006418:Heme; D008345:Manganese; D006639:Histidine; D003576:Electron Transport Complex IV