REACTION OF CYTOCHROME BO3 WITH OXYGEN - EXTRA REDOX CENTER(S) ARE PRESENT IN THE PROTEIN

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.