The reaction of hydrogen peroxide with pulsed cytochrome bo from Escherichia coli.
The reaction of hydrogen peroxide (H2O2) with pulsed cytochrome bo leads to characteristic spectral changes in the enzyme. The difference spectrum shows minima at 401, 494 and 628 nm, and maxima at 420, approximately 468, 526 and 556 nm. delta epsilon 420-epsilon 401 is in the range 73-86 mM-1.cm-1 and delta epsilon 556-epsilon 628 is 7.7-9.6 mM-1.cm-1 (taking delta epsilon 560-epsilon 580 for the reduced minus oxidised spectrum to be 20.5 mM-1.cm-1). The stoichiometry of the reaction, determined by titration of the spectral changes, is 1:1. The second order rate constant for the reaction, which is 1.0-1.5 x 10(3) M-1.s-1 at 20 degrees C, is independent of pH over the range 6.5-8.0. The product of the reaction decays with a first-order rate constant in the range 1-4 x 10(-4) s-1, so the Kd value is apparently in the range 0.05-0.40 microM. The spectral changes observed immediately after quinol-induced turnover, or during steady-state turnover induced by hydrazine or by carbon monoxide, are qualitatively the same as those induced by H2O2 though of lower amplitude. H2O2 addition perturbs the hydrazine-induced or CO-induced steady states by increasing the amplitude of the spectral changes, but there is no qualitative change. From this observation, and the 1:1 stoichiometry of the reaction, we conclude that the intermediate induced by H2O2, which we term F., requires donation of only two electrons to the enzyme from an external source.
Moody
A J
AJ
Glynn Research Institute, Bodmin, Cornwall, England.
Rich
P R
PR
D002248:Carbon Monoxide / Q000494:pharmacology; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006834:Hydrazines / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010100:Oxygen / Q000494:pharmacology; D013053:Spectrophotometry
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D006834:Hydrazines; C029424:hydrazine; D002248:Carbon Monoxide; C017168:cytochrome bo, E coli; D006861:Hydrogen Peroxide; D010100:Oxygen