Reaction of Cytochrome bo₃, with Oxygen: Extra Redox Center(s) are Present in the Protein

The reaction of oxygen with cytochrome bo₃, a quinol oxidase from Escherichia coli, has been studied by resonance Raman scattering after initiation of the reaction by CO photolysis in a continuous flow apparatus and by directly mixing the enzyme with oxygen. The high-frequency region of the spectrum was monitored to determine the time evolution of the spin, oxidation, and coordination states of heme O and the oxidation 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 75µs, O₂ reacted with heme O and formed a low-spin ferric or ferryl adduct without significant oxidation of heme B. In phase II, between 75 and 120 µs, a small fraction of heme B was oxidized. In phase III, at ~1 s, the majority of heme B was oxidized and heme O reverted to a high-spin ferric state. The high rate of oxygen reduction 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 our enzyme preparations rule out a quinol in the tight binding (Q_H) site as a possible donor but instead suggest electron donation from the protein matrix, such as from tryptophans or tyrosines. Three Tryptophans (W280, W282, and W331) and one tyrosine (Y288) are postulated as candidates for such a role, and their location near the binuclear center suggests that the donor electrons follow a pathway directly to the heme O-Cu_B binuclear center without passing through heme B. The donors that participate in the catalytic mechanism in vitro may also play a functional role under physiological conditions.