Jl. Wang et al., REACTION OF CYTOCHROME BO3 WITH OXYGEN - EXTRA REDOX CENTER(S) ARE PRESENT IN THE PROTEIN, Biochemistry, 34(47), 1995, pp. 15504-15511
The reaction of oxygen with cytochrome bo(3), a quinol oxidase from Es
cherichia coli, has been studied by resonance Raman scattering after i
nitiation of the reaction by CO photolysis in a continuous flow appara
tus and by directly mixing the enzyme with oxygen. The high-frequency
region of the spectrum was monitored to determine the time evolution o
f the spin, oxidation, and coordination states of heme O and the oxida
tion state of heme B by using newly established marker lines for each
heme. Three phases of the reaction were detected. In phase I, complete
in 75 mu s, O-2 reacted with heme O and formed a low-spin ferric or f
erryl adduct without significant oxidation of heme B. In phase II, bet
ween 75 and 120 mu s, a small fraction of heme B was oxidized. In phas
e III, at similar to 1 s, the majority of heme B was oxidized and heme
O reverted to a high-spin ferric state. The high rate of oxygen reduc
tion at heme O to the three- or four-electron reduced level, despite a
very low rate of heme B oxidation, indicates that there are electron
donors active in the enzyme other than the metal centers. Assays of ou
r enzyme preparations rule out a quinol in the tight binding (Q(H)) Si
te as a possible donor but instead suggest electron donation from the
protein matrix, such as from tryptophans or tyrosines. Three Tryptopha
ns (W280, W282, and W331) and one tyrosine (Y288) are postulated as ca
ndidates for such a role, and their location near the binuclear center
suggests that the donor electrons follow a pathway directly to the he
me O-Cu-B binuclear center without passing through heme B. The donors
that participate in the catalytic mechanism in vitro may also play a f
unctional role under physiological conditions.