Direct measurement by laser flash photolysis of intramolecular electron transfer in the three-electron reduced form of ascorbate oxidase from zucchini.

Ascorbate oxidase, which has been fully reduced by its substrate, can rapidly transfer a single electron to the laser-generated triplet state of 5-deazariboflavin. Subsequent to this, intramolecular electron transfer occurs resulting in the oxidation of the blue type I copper center. This latter process proceeds via biphasic kinetics, with observed rate constants of 9500 s-1 and 1400 s-1, both of which are protein concentration independent. This indicates that the initial oxidation reaction involves the type II, III trinuclear center, probably occurring via parallel reactions of two of the three copper atoms. The rate constants for intramolecular electron transfer in the three-electron reduced enzyme are one to two orders of magnitude larger than previously observed for the one-electron reduced enzyme, indicating a dramatic effect of the redox state of the enzyme on the intramolecular communication between the copper centers.