title: ENDOR and ESEEM studies of cytochrome c oxidase: evidence for exchangeable protons at the CuA site.
abstract: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were used to study whether protons in the immediate protein environment around CuA in cytochrome c oxidase are susceptible to solvent exchange. The enzyme was incubated in buffered D2O under resting or turnover conditions for 90 min and then frozen to quench the hydrogen/deuterium-exchange process. ENDOR spectra of the deuterated sample were essentially identical to those of control samples. The ESEEM spectra, however, provided a clear indication of the introduction of deuterium into the CuA environment following incubation in buffered D2O. The extent of deuterium incorporation was not affected by enzyme turnover. An analysis of the ESEEM data indicated that water is in reasonably close proximity to the CuA site, but not in the immediate coordination sphere of the metal(s). We estimate a minimum distance of 5.4 A between the CuA center and the protein/water interface. This relatively short surface separation distance is consistent with the role of CuA as the immediate oxidant of cytochrome c in the cytochrome oxidase (Hill, B. C. (1991) J. Biol. Chem. 266, 2219-2226).
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing 48824.
authors.forename: A P
authors.initials: AP
authors.lastname: Hansen
authors.forename: R D
authors.initials: RD
authors.lastname: Britt
authors.forename: M P
authors.initials: MP
authors.lastname: Klein
authors.forename: C J
authors.initials: CJ
authors.lastname: Bender
authors.forename: G T
authors.initials: GT
authors.lastname: Babcock
mesh_terms: D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003300:Copper / Q000378:metabolism*; D003903:Deuterium; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D014867:Water / Q000378:metabolism
chemical_list: D011522:Protons; D014867:Water; D003300:Copper; D003903:Deuterium; D003576:Electron Transport Complex IV