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

• Published in: Biochemistry (Easton) 1995-11, Vol.34 (47), p.15504-15511
• Main Authors: Wang, Jianling, Rumbley, Jon, Ching, Yuan-chin, Takahashi, Satoshi, Gennis, Robert B, Rousseau, Denis L
• Format: Article
• Language: English
• Subjects: Computer Simulation; Cytochromes - chemistry; Cytochromes - metabolism; Escherichia coli - enzymology; Escherichia coli Proteins; Oxidation-Reduction; Oxygen - metabolism; Spectrum Analysis, Raman
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00047a016

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 75 microseconds, 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 microseconds, a small fraction of heme B was oxidized. In phase III, at approximately 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 (QH) site as a possible donor but instead suggest electron donation from the protein matrix, such as from tryptophans or tyrosine.