Hydrogen peroxide induced spectral change in resting cytochrome oxidase and the ph effect
Previous studies of hydrogen peroxide interaction with oxidized cytochrome oxidase showed a lack of uniformity and consistency. Problems arose from the use of heterogeneous enzyme (isolated at pH 7.0-7.4) and a large excess of hydrogen peroxide. This study differs in two principle ways: first, homogeneous resting enzyme purified under alkaline conditions was used and experimental conditions were controlled in the range of pH 8.6-9.2 allowing the elimination of the interference of heterogeneity of the enzyme; second, peroxide concentration was kept at a relatively low level to avoid complications caused by a large excess of peroxide. Hydrogen peroxide induced spectroscopic changes in resting cytochrome oxidase was investigated in the Soret and visible regions. The results showed a homogeneous response in the Soret and the 655 nm charge transfer band regions. The Soret band intensified and shifted from 423 to 428 nm with a single, rapid exponential binding reactivity at 435 and 655 nm. However, the spectral response to peroxide was heterogeneous at the α - and β-bands in the visible region. An increase in amplitude of at 606 nm was associated with a red-shift of the α-band peak from 598 to 601 nm, producing which sequently decayed, causing the a-band to blue shift back to approximately the original position. An approximate isosbestic point at 588 nm suggests that the decay of the 606 nm band correlates with an increase in absorbance at 580 nm. Peroxide-binding kinetics at 606 nm explained by two sequential, reversible peroxide concentration-dependent steps at pH 9.0 with average bimolecular rate constants of 391 and 38 M⁻¹∙s⁻¹ and equilibrium dissociation constants of 5.1 and 20 pM, respectively. pH effects of peroxide interaction with resting cytochrome oxidase was also investigated in this study. A two-fold of change in the bimolecular rate constants was induced by a two-fold of change in pH. The weak pH effects probably reflect a change in the diffusion of peroxide to the binding site rather than an effect on the bind site. Results were consistent with earlier pH studies. Two principle conclusions are made: first, resting cytochrome oxidase purified at high pH is structurally similar to the pulsed enzyme; second, the stability of the Soret band during the decay of the 606 nm band indicates a chemical species other than cytochrome a₃ is involved. The decay at 606 nm is proposed to result from quenching of an amino acid radical, possibly associated with Cug, analogous to the conversion of cytochrome c peroxidase compound I into compound II.