Resonance Raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide

Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O-2 by quinols and simultaneously translocates protons across the cytoplasmic membrane. The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system. Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm(-1) for intermediates derived from (H2O2)-O-16/(H2O2)-O-18. The experiments using (H2OO)-O-16-O-18 yielded no new bands, indicating that all the bands arose from the Fe=O stretching (nu(Fe=O)) mode. Among them, the intensity of the 805/X cm(-1) pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome be appeared prior to the formation of the F species at higher pH. For this intermediate, a Raman band assignable to the C-O stretching mode of a tyrosyl radical was deduced at 1489 cm(-1) from difference spectra. This suggests that the P intermediate of cytochrome bo contains an Fe-IV=O heme and a tyrosyl radical like compound I of prostaglandin H synthase. The 783/753 cm(-1) pair, which was dominant at neutral pH and close to the nu(Fe=O) frequency of the oxofenyl intermediate of CcO, presumably arises from the F intermediate. On the contrary, the (767)/730 cm(-1) species has no counterpart in CcO, Its presence may support the branched reaction scheme proposed previously for O-2 reduction by cytochrome bo.