DIRECT MEASUREMENT BY LASER FLASH-PHOTOLYSIS OF INTRAMOLECULAR ELECTRON-TRANSFER IN THE 3-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 stat e of 5-deazariboflavin. Subsequent to this, intramolecular electron tr ansfer occurs resulting in the oxidation of the blue type I copper cen ter. This latter process proceeds via biphasic kinetics, with observed rate constants of 9500 s(-1) and 1400 s(-1), both of which are protei n concentration independent. This indicates that the initial oxidation reaction involves the type II, III trinuclear center, probably occurr ing via parallel reactions of two of the three copper atoms. The rate constants for intramolecular electron transfer in the three-electron r educed enzyme are one to two orders of magnitude larger than previousl y observed for the one-electron reduced enzyme, indicating a dramatic effect of the redox state of the enzyme on the intramolecular communic ation between the copper centers.