Inhibition of intramolecular electron transfer in ascorbate oxidase by Ag+: redox state dependent binding
详细信息 查看全文
- 作者：Santagostini ; Laura ; Gullotti ; Michele ; Hazzard ; James T. ; Maritano ; Silvana ; Tollin ; Gordon ; et. al.
- 关键词：Ascorbate oxidase ; Silver ; Enzyme inhibition ; Electron transfer ; Laser flash photolysis
- 刊名：Journal of Inorganic Biochemistry
- 出版年：2005
- 出版时间：February, 2005
- 年：2005
- 卷：99
- 期：2
- 页码：600-605
- 全文大小：160 K
文摘
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??108 M???1s???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.].