Inhibition of intramolecular electron transfer in ascorbate oxidase by Ag+: redox state dependent binding

Intramolecular electron transfer within zucchini squash ascorbate oxidase is inhibited in a novel manner in the presence of an equimolar concentration of Ag(+). At pH 5.5 in acetate buffer reduction of the enzyme by laser flash photolytically generated 5-deazariboflavin semiquinone occurs at the Type I Cu with a rate constant of 5 x 10(8) M(-1)s(-1). Subsequent to this initial reduction step, equilibration of the reducing equivalent between the Type I Cu and the trinuclear Type II, III copper cluster (TNC) occurs with rate constant of 430 s(-1). The 41% of the reduced Type I Cu is oxidized by this intramolecular electron transfer reaction. When these reactions are performed in the presence of Ag(+) equimolar to dimeric AO, the bimolecular reduction of the enzyme by the 5-deazariboflavin semiquinone is not affected. As in the case of the native enzyme, intramolecular electron transfer between the Type I Cu and the TNC occurs, which continues until 25% of the reducing equivalent has been transferred. At that point, the reducing equivalent is observed to more slowly return to the Type I Cu, resulting a second reduction phase whose rate constant (100 s(-1)) is protein and Ag(+) concentration independent. The data suggest that partial reduction of the TNC results in Ag(+) binding to the enzyme which causes the apparent midpoint potential of the TNC as a whole to decrease thereby reversing the direction of electron flow. These results are consistent with the inhibitory effect of Ag(+) on the steady-state activity of ascorbate oxidase [S. Maritano, E. Malusa, A. Marchesini, presented at The Meeting on Metalloproteins, SERC Daresbury Laboratory, Warrington, England, 1992; A. Marchesini, XIX Convegno Nazionale SICA, Italian Society of Agricultural Chemistry, Reggio Calabria, Italy, September 2001.].