DISTINCT ROLES OF 2 HEME CENTERS FOR TRANSMEMBRANE ELECTRON-TRANSFER IN CYTOCHROME B(561) FROM BOVINE ADRENAL CHROMAFFIN VESICLES AS REVEALED BY PULSE-RADIOLYSIS

The reaction of monodehydroascorbate (MDA) radical with purified cytoc hrome b(561) from bovine adrenal chromaffin vesicles was investigated by the technique of pulse radiolysis. Radiolytically generated MDA rad ical oxidized rapidly the reduced form of cytochrome b(561) to yield t he oxidized form. Subsequently the oxidized form of cytochrome b(561) was re-reduced by ascorbate in the medium. The second-order rate const ants of the reaction of MDA radical were increased with decreasing pH, whereas a maximum of the second-order rate constant for the reaction with ascorbate was obtained around pH 6.8. At excess MDA radical to cy tochrome b(561) concentration, only half of the heme in cytochrome b56 1 was oxidized, indicating that only one of the two heme centers can r eact with MDA radical. On the other hand, when the reactions were exam ined using cytochrome b(561) pretreated in a mild alkaline condition i n the oxidized state, the cytochrome b(561) could not be oxidized with MDA radical, suggesting that the heme center specific for the electro n donation to MDA radical is selectively modified upon the alkaline tr eatment. These results suggest that the two heme b centers have distin ct roles for the electron donation to MDA radical and the electron acc eptance from ascorbate, respectively.