Yc. Fann et al., 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, Biochemistry, 34(32), 1995, pp. 10245-10255
We have studied the structure of the Cu-B site in the binuclear heme-c
opper center of the fully oxidized form of the quinol-oxidizing cytoch
rome aa(3)-600 from Bacillus subtilis by EXAFS and ENDOR spectroscopy.
This enzyme is member of the large superfamily of heme-copper respira
tory oxidases, which catalyze the reduction of dioxygen to water and l
ink it to translocation of protons across the bacterial or mitochondri
al membrane. The EXAFS of the Cu-B site strongly suggests tetragonal c
oordination by two or three histidines with one or two O/N donor ligan
ds. There are some indications that a Cl- ion might fractionally occup
y 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 spectrosco
py of the Cu-B site in (H2O)-H-1 and (H2O)-H-2 media showed evidence o
f an oxygenous copper ligand with an exchangeable proton. N-14 ENDOR r
evealed three inequivalent nitrogenous ligands with hyperfine coupling
constants consistent with histidines. Together, these results strongl
y suggest that the fully oxidized enzyme has a low-symmetry, tetragona
l 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.