Cytochrome c peroxidase-catalyzed oxidation of yeast iso-1 ferrocytochrome c by hydrogen peroxide. Ionic strength dependence of the steady-state parameters

Abstract

The cytochrome c peroxidase-catalyzed oxidation of yeast iso-1 ferrocytochrome c by hydrogen peroxide can be understood on the basis of a mechanism involving two cytochrome c-binding sites on cytochrome c peroxidase. Values of the equilibrium dissociation constants for both the high- and low-affinity binding sites determined from the steady-state kinetic measurements agree well with published values obtained by vastly different techniques, providing strong support for the two-binding site mechanism. Maximum enzyme turnover via oxidation of cytochrome c bound at the high-affinity site increases from 2 to 860 s-1 as the ionic strength is increased from 0.010 to 0.20 M. Oxidation of yeast iso-1 ferrocytochrome c is faster in the 2:1 complexes of cytochrome c peroxidase compounds I and II in comparison to the 1:1 complexes. The oxidation rates in the 2:1 complex are macroscopic rate constants equal to the sum of the oxidation rates via both the high- and low-affinity sites. The maximum enzyme turnover via the 2:1 complex increases from 1100 to 2700 s-1 over the ionic strength range 0.010-0.070 M.

Cobalamin Protection against Oxidative Stress in the Acidophilic Iron-oxidizing Bacterium Leptospirillum Group II CF-1

“…The absorbance of a ferricyanide-oxidized cytochrome c was also determined. The percentage of cytochrome c reduction was estimated according to Matthis and Erman (1995) using an extinction coefficient (𝜀) of 27.7 mM -1 cm -1 . To measure CcP activity, the reaction mixture (500 μL) contained 10 mM potassium phosphate pH 7.0, 25 mM ferrocytochrome c , and 50 μg protein extract.…”

Charge Reversal of a Critical Active‐Site Residue of Cytochrome‐c Peroxidase

“…Hydrogen peroxide solutions were standardized by titration with potassium permanganate [29]. Steady-state rates of the cytochrome-c peroxidase-catalyzed oxidation of yeast ferrocytochrome c by H,O, were determined as described by Kang analyzed at pH 6.2 using a saturating concentration of hydrogen peroxide (200 pM) [30]. Under the high ionic strength conditions of the assay, the data could be fitted to an equation describing a single saturable process 1301:…”

Characterization of Four Covalently-Linked Yeast Cytochrome c/Cytochrome c Peroxidase Complexes: Evidence for Electrostatic Interaction between Bound Cytochrome c Molecules

“…(open squares) obtained between 1.8 mM and 118 mM ionic strength, between pH 6 and 7, using proton uptake measurements (11) and dynamic fluorescence quenching of zinc-modified CcP (7,14,15). Michaelis constants for the lowaffinity phase, K M2 (open diamonds), of the steady-state oxidation of yeast iso-1 ferrocytochrome c catalyzed by yCcP (19) and CcP(MI) (23) are included. Microscopic model for the binding of cytochrome c to two unique sites on CcP, a primary binding site (right-hand side) and a secondary binding site (left-hand side).…”