The Room Temperature Reaction of Carbon Monoxide and Oxygen with the Cytochrome bd Quinol Oxidase from Escherichia Coli

Bruce C. Hill, John J. Hill, Robert B. Gennis

Research output: Contribution to journalArticlepeer-review

Abstract

When grown under O2-limited conditions, Escherichia coli expresses a cytochrome bd quinol oxidase that has an unusually high affinity for O2. We have studied the reaction of cytochrome bd with CO and O2 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 × 107 M-1 s-1. Reaction of CO-bound enzyme with O2 gives a CO off-rate of 1.6 s-1. The O2 reaction is followed by a flow–flash procedure in which CO-ligated enzyme is mixed with O2, and the reaction commenced by photolysis of cytochrome d–CO. In the presence of O2, two processes are resolved on a time—scale of 300 μs. The absorbance at 645 nm first increases at a rate that is dependent on O2 concentration with a value of 2 × 109 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 O2 concentration above 50 μ O2 and reaches a first-order limit of 1 × 104 s-1. A model for the reaction of the cytochrome bd quinol oxidase with O2 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 time as the ferryl species forms. We suggest that the suitability of the cytochrome bd quinol oxidase to function at low O2 concentration is conferred by its rapid rate of binding O2.

Original languageEnglish (US)
Pages (from-to)15110-15115
Number of pages6
JournalBiochemistry
Volume33
Issue number50
DOIs
StatePublished - Dec 1 1994

ASJC Scopus subject areas

  • Biochemistry

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