Structure of CuB in the Binuclear HemeŃCopper Center of the Cytochrome aa3-Type Quinol Oxidase from Bacillus subtilis: An ENDOR and EXAFS Study.

Yang C. Fann, Ishak Ahmed, Ninian J Blackburn, John S. Boswell, Marina L. Verkhovskaya, Brian M. Hoffman, and Marten Wikstrom. Biochemistry 34 10245-10255 (1995)

Summary>. We have studied the structure of the CUB site in the binuclear hemeŃcopper center of the fully oxidized form of the quinol-oxidizing cytochrome aa3-600 frorn Bacillus subtilis by EXAFS and ENDOR spectroscopy. This enzyme is member of the large superfamily of hemeŃcopper respiratory oxidases, which catalyze the reduction of dioxygen to water and link it to translocation of protons across the bacterial or mitochondrial membrane. The EXAFS of the CuB site strongly suggests tetragonal coordination by two or three histidines with one or two O/N donor ligands. There are some indications that a C1- ion might fractionally occupy substitution-labile sites, although the majority of enzyme molecules did not contain any heavy (second row) scatterers, indicative of a C1- (or S) bridge between the heme iron and CUB [cf. Powers, L., et al. (1994) Biochim. Biophys. Acta 1183. 504Ń512]. Proton ENDOR spectroscopy of the CuB site in H20 and 2HrO media showed evidence of an oxygenous copper ligand with an exchangeable proton. 14N ENDOR revealed three inequivalent nitrogenous ligands with hyperflne coupling constants consistent with histidines. Together, these results strongly suggest that the fully oxidized enzyme has a low-symmetry, tetragonal CuB site with three histidine ntrro˘ens and one oxygen as ligands, the latter with an exchangeable protons). The identity and assignment of these ligands are discussed