T. Uchida et al., Resonance Raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide, BIOCHEM, 39(22), 2000, pp. 6669-6678
Cytochrome bo from Escherichia coli, a member of the heme-copper terminal o
xidase superfamily, physiologically catalyzes reduction of O-2 by quinols a
nd 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 microcircu
lating 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 ban
ds, 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 high
er 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 re
ported 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 ban
d 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 compo
und I of prostaglandin H synthase. The 783/753 cm(-1) pair, which was domin
ant at neutral pH and close to the nu(Fe=O) frequency of the oxofenyl inter
mediate 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 s
upport the branched reaction scheme proposed previously for O-2 reduction b
y cytochrome bo.