Reaction of hydrogen peroxide with the rapid form of resting cytochrome oxidase.
The hydrogen peroxide binding reaction has been examined with alkaline-purified resting enzyme in order to avoid mixtures of low pH induced fast and slow conformers. At pH 8.8-9.0 (20 degrees C), the reactivity of resting enzyme was similar to the peroxide-free, pulsed conformer that has been characterized by other investigators. The reaction showed single-phase reactivity at 435 and 655 nm and required a minimum 8:1 molar excess of peroxide (over cytochrome a3) for quantitative reaction. At 16:1, the Soret band was stable for 1.0-1.5 h, but above 80:1, the band began showing generalized attenuation within 1-2 min. The peroxide binding reaction was also associated with an increase in absorbance at 606 nm which correlated with the rate of change at 435 and 655 nm. The observed rate constants at each of these wavelengths showed similar linear dependence on peroxide concentration, giving an average bimolecular rate constant of 391 M-1.s-1 and a Kd of 5.1 microM. The rise phase at 606 nm was observed to saturate at an 8:1 molar excess of peroxide but showed a slow, concentration-dependent first-order decay that gave a bimolecular rate constant and Kd of 38 M-1.s-1 and 20 microM, respectively. The decay was not associated with a change in the Soret absorption or charge-transfer regions, suggesting a type of spectral decoupling. An isosbestic point at 588 nm was consistent with the 606- to 580-nm conversion proposed by other investigators, although direct observation of a new band at 580 nm was difficult.(ABSTRACT TRUNCATED AT 250 WORDS)
Weng
L C
LC
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Baker
G M
GM
D001665:Binding Sites / Q000187:drug effects; D003486:Cyanides; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D010084:Oxidation-Reduction / Q000187:drug effects; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D003486:Cyanides; D008024:Ligands; D006861:Hydrogen Peroxide; D003576:Electron Transport Complex IV