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.].
Santagostini
Laura
L
Dipartimento di Chimica Inorganica, Metallorganica e Analitica, Istituto ISTM, Universitá di Milano, Via Venezian 21, 20133 Milano, Italy. laura.santagostini@unimi.it
Gullotti
Michele
M
Hazzard
James T
JT
Maritano
Silvana
S
Tollin
Gordon
G
Marchesini
Augusto
A
D001204:Ascorbate Oxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D028464:Cucurbita / Q000201:enzymology; D004579:Electron Transport / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D010782:Photolysis; D012834:Silver / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D012834:Silver; D003300:Copper; D001204:Ascorbate Oxidase