Electron-transfer from cytochrome c to ascorbate oxidase and its type 2 copper-depleted derivatives

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting cucumber ascorbate oxidase as functions of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 820 M-1 s-1 in 0.2 M phosphate buffer at pH 6.0 and 25 degrees C. The activation parameters were estimated to be delta H++ = 5 kJ mol-1 and delta S++ = -188 Jmol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that the electron-transfer from cytochrome c to ascorbate oxidase is realized by the local electrostatic interaction between them in spite of the reaction between positively charged proteins. Reactions of type 2 copper-depleted ascorbate oxidase whose type 3 coppers were in the reduced or oxidized form indicated that the type 1 copper site accepts an electron from cytochrome c. The reaction rate was remarkably increased with decreasing pH for both the native enzyme and derivatives. Further, on addition of hexametaphosphate anion the rate of the electron-transfer decreased because the association of both proteins to realize the electron-transfer was inhibited due to a change in distribution of the local charge on the protein surface(s).