STRUCTURE OF CU-B IN THE BINUCLEAR HEME-COPPER CENTER OF THE CYTOCHROME AA(3)-TYPE QUINOL OXIDASE FROM BACILLUS-SUBTILIS - AN ENDOR AND EXAFS STUDY

We have studied the structure of the Cu-B site in the binuclear heme-copper center of the fully oxidized form of the quinol-oxidizing cytochrome aa(3)-600 from Bacillus subtilis by EXAFS and ENDOR spectroscopy. This enzyme is member of the large superfamily of heme-copper 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 Cu-B site strongly suggests tetragonal coordination by two or three histidines with one or two O/N donor ligands. There are some indications that a Cl- ion might fractionally occupy substitution-labile sites, although the majority of enzyme molecules did not contain any heavy (second row) scatterers, indicative of a Cl- (or S) bridge between the heme iron and Cu-B [cf. Powers, L., et al. (1994) Biochim. Biophys. Acta 1183, 504-512]. Proton ENDOR spectroscopy of the Cu-B site in (H2O)-H-1 and (H2O)-H-2 media showed evidence of an oxygenous copper ligand with an exchangeable proton. N-14 ENDOR revealed three inequivalent nitrogenous ligands with hyperfine coupling constants consistent with histidines. Together, these results strongly suggest that the fully oxidized enzyme has a low-symmetry, tetragonal Cu-B site with three histidine nitrogens and one oxygen as ligands, the latter with an exchangeable proton(s). The identity and assignment of these ligands are discussed.