Specific ion effects on the reaction between cytochrome c peroxidase and hydrogen peroxide
The second-order reaction between cytochrome c peroxidase and hydrogen peroxide to produce compound I is subject to large specific ion effects below pH 6. If the reaction is carried out in buffers containing 90 mM potassium nitrate, the bimolecular rate constant is pH dependent, decreasing at low pH with an apparent pKg of 5.5. In 100 mM phosphate buffers the pKₐ shifts to less than 4.5. At pH 4.5, the bimolecular rate constant is about 35 /μM⁻¹s⁻¹ for buffers containing 90 to 100 mM phosphate or sulfate. The rate constant decreases to 31, 28, 6, and 5 /μM⁻¹s⁻¹ in buffers containing 90 mM MES, acetate, nitrate, and chloride, respectively. At pH 4.5, the apparent bimolecular rate constant decreased with increasing concentrations of nitrate ion in a 10 mM acetate buffer. The ionic strength was held constant at 0.1 M by using phosphate to compensate for changes in the nitrate concentration. The data plural strongly support a model in which nitrate ion binds to the enzyme and inhibits the hydrogen peroxide reaction. When cytochrome c peroxidase is mixed with hydrogen peroxide to form compound I, a minor, hydrogen peroxide-independent reaction is often observed, especially below pH 6. The amplitude of the reaction is generally small, averaging about 11% of the total reaction amplitude in these studies.