Reaction of Ascorbic Acid with Cytochrome

Rate constants for reduction of cytochrome bsel by internal ascorbate (kOA) and oxidation by external ferricyanide (klF) were determined as a function of pH from rates of steady-state electron transfer across chromaffin-vesicle membranes. The pH dependence of electron transfer from cytochrome b56l to ferricyanide (klF) may be attributed to the pH dependence of the membrane surface potential. The rate constant for reduction by internal ascorbate (koA), like the previously measured rate constant for reduction by external ascorbate ( k l A ) , is not very pH-dependent and is not consistent with reduction of cytochrome beG1 by the ascorbate dianion. The rate at which ascorbate reduces cytochrome b56, is orders of magnitude faster than the rate at which it reduces cytochrome c, despite the fact that midpoint reduction potentials favor reduction of cytochrome c. Moreover, the rate constant for oxidation of cytochrome bsal by ferricyanide (klF) is smaller than the previously measured rate constant for oxidation by semidehydroascorbate, despite the fact that ferricyanide has a higher midpoint reduction potential. These results may be reconciled by a mechanism in which electron transfer between cytochrome and ascorbate/semidehydroascorbate is accelerated by concerted transfer of a proton. This may be a general property of biologically significant electron transfer reactions of ascorbic acid.