Kinetics of electron and proton transfer during the reaction of wild type and helix VI mutants of cytochrome bo3 with oxygen.
Site-directed mutagenesis was used to investigate the mechanism of electron and proton transfer in the ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The reaction between the fully reduced form of the enzyme and dioxygen was studied using the flow--flash method. After rapid mixing of CO-bound enzyme with an O2-containing solution, CO was photodissociated, and the subsequent electron- and proton-transfer reactions were measured spectrophotometrically, the latter using a pH-indicator dye. In the wild-type, pure bo3 enzyme, without bound quinones, we observed a single kinetic phase with a rate constant of about 2.4 x 10(4) s-1, associated with formation of the ferry1 oxygen intermediate, followed by proton uptake from solution with a rate constant of about 1.2 x 10(4) s-1. Enzyme in which heme o instead of heme b was incorporated into the low-spin site displayed a slower ferry1 formation with a rate constant of about 3.6 x 10(3) s-1. Upon replacement of the acidic residue glutamate 286 in helix VI of subunit I with a nonprotonatable residue, electron transfer was slightly accelerated, and proton uptake was impaired. Mutations of other residues in the vicinity of E286 also resulted in a dramatic decrease of proton uptake, suggesting that the environment of this residue is important for efficient proton transfer. In the closely related cytochrome aa3 from P. denitrificans, the corresponding residue (E278) has been suggested to be part of a proton-transfer pathway [Iwata, S., Ostermeier, C., Ludwig, B., & Michel, H. (1995) Nature 376, 660-669]. The results are discussed in terms of a model for electron-proton coupling during dioxygen reduction.
Svensson-Ek
M
M
Department of Biochemistry and Biophysics, University of Göteborg, Sweden.
Thomas
J W
JW
Gennis
R B
RB
Nilsson
T
T
Brzezinski
P
P
D002248:Carbon Monoxide / Q000378:metabolism; D003001:Cloning, Molecular; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010782:Photolysis; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D011522:Protons; D011994:Recombinant Proteins; C083850:cytochrome bo3, E coli; D014451:Ubiquinone; D002248:Carbon Monoxide; C003741:ubiquinol; D010100:Oxygen