Oxygen reaction and proton uptake in helix VIII mutants of cytochrome bo3.
The oxygen reaction of wild-type and helix VIII mutants of cytochrome bo3 from Escherichia coli, and the associated proton uptake during this reaction, has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen. We have focused on mutations in the transmembrane helix VIII where protonatable residues have been exchanged, and mainly on the inactive mutants (i.e., T352A, T359A, and K362L, -M, and -Q). The kinetics for electron transfer during oxidation for the mutants are similar to the wild-type; two rate constants of 3.2 x 10(4) and 3.4 x 10(3) s-1 (at 1 mM oxygen) are detected. Proton uptake is observed for wild-type as well as for the mutant enzymes, but the mutations within helix VIII have affected the rate of proton uptake; it is significantly accelerated in the mutants. These results show that none of the protonatable residues in helix VIII are required in the reaction between the fully reduced cytochrome bo3 and oxygen. We have also studied electron redistribution after photolysis of CO from the mixed-valence compound; we found three kinetic components for wild-type and the mutants T352A and T359A, but for K362M only the first and third components are observed, with amplitudes that are lower than those for the corresponding components in the wild-type enzyme, suggesting that the characteristics of internal electron transfer in the K362M mutant are different from those of the wild-type enzyme.
Svensson
M
M
Department of Biochemistry and Biophysics, Göteborg University, Sweden.
Hallén
S
S
Thomas
J W
JW
Lemieux
L J
LJ
Gennis
R B
RB
Nilsson
T
T
D002248:Carbon Monoxide / Q000737:chemistry; D002384:Catalysis; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010782:Photolysis; D017433:Protein Structure, Secondary; D011522:Protons
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D011522:Protons; C083850:cytochrome bo3, E coli; D002248:Carbon Monoxide