PEROXY AND FERRYL INTERMEDIATES OF THE QUINOL-OXIDIZING CYTOCHROME AA3 FROM BACILLUS-SUBTILIS

The quinol-oxidizing cytochrome aa3-600 from Bacillus subtilis has a binuclear heme a3-Cu(B) center of O2 reduction and a low-spin heme a, but lacks a fourth redox center, Cu(A), which is a typical component of cytochrome c oxidases. Fully reduced (3e-) cytochrome aa3-600 and the two-electron-reduced CO complex were allowed to react with O2 at 0-degrees-C, and the reaction products were studied by optical spectroscopy. When the two-electron-reduced CO complex (heme a3 and Cu(B) are reduced, but the low-spin heme is oxidized) reacts with O2 at neutral pH, a compound is produced that may be assigned a ferric-cupric peroxy structure (P). At low pH, this species spontaneously decomposes into another compound, which may be assigned a ferryl structure (F). When fully reduced enzyme (3e-) reacts with O2 at high pH, a peroxy species is the primary product. This subsequently decays into F, followed by very slow decay of the latter. Our data show that at high pH the third electron, which is required to convert P into F, resides for a relatively long time in either Cu(B) or heme a3. This suggests that transfer of the third electron to the binuclear center is followed by proton uptake, which must occur before scission of the O-O bond. The present data strongly support the involvement of discrete peroxy and ferryl intermediates in the catalytic cycle of cytochrome aa3-600. The dioxygen reduction mechanism in the binuclear site is thus very similar in the quinol and the cytochrome c oxidases.