THE ROOM-TEMPERATURE REACTION OF CARBON-MONOXIDE AND OXYGEN WITH THE CYTOCHROME BD QUINOL OXIDASE FROM ESCHERICHIA-COLI

When grown under O-2-limited conditions, Escherichia coli expresses a cytochrome bd quinol oxidase that has an unusually high affinity for O-2. We have studied the reaction of cytochrome bd with CO and O-2 by rapid-reaction spectrophotometry. The reduced enzyme forms a photosensitive ferrocytochrome d-CO complex, and following photolysis, CO recombines with the reduced enzyme with a bimolecular rate of 8 x 10(7) M(-1) s(-1). Reaction of CO-bound enzyme with O-2 gives a CO off-rate of 1.6 s(-1). The O-2 reaction is followed by a flow-flash procedure in which CO-ligated enzyme is mixed with O-2, and the reaction commenced by photolysis of cytochrome d-CO. In the presence of O-2, two processes are resolved on a time-scale of 300 mu s. The absorbance at 645 nm first increases at a rate that is dependent on O-2 concentration with a value of 2 x 10(9) M(-1) s(-1). The second phase results in decreased absorbance at 645 nm and increased absorbance at 680 nm. The rate of the second process is independent from O-2 concentration above 50 mu M O-2 and reaches a first-order limit of 1 x 10(4) s(-1). A model for the reaction of the cytochrome bd quinol oxidase with O-2 is proposed in which an initial ferrocytochrome d-oxy adduct forms, and then decays to a ferryl-oxo species. The oxidation of the low-spin cytochrome b component of the oxidase, monitored at 560 nm, occurs at the same lime as the ferryl species forms. We suggest that the suitability of the cytochrome bd quinol oxidase to function at low O-2 concentration is conferred by its rapid rate of binding O-2.