Kinetics of Trolox C oxidation by lactoperoxidase compound II

The lactoperoxidase (LPO) compound II catalyzed oxidation of Trolox C, a vitamin E water-soluble derivative, was studied by rapid scan spectral analysis and stopped-flow kinetic measurements. Our rapid scan spectral analysis clearly indicates that LPO compound II is reduced to native enzyme by Trolox C; hence the reaction is a one-electron redox process. The reaction was investigated at pH's ranging from 3.0 to 7.0. Trolox C is more reactive with LPO-II in acidic solutions. Kinetic and spectroscopic studies demonstrate that LPO has a binding site in the vicinity of the heme for Trolox C. Trolox C exhibits a quantitative 1:1 binding to native LPO in acidic solutions. The binding ability of Trolox C to native LPO decreases with increasing pH. The same trend is observed when the second order rate constants kapp for the reaction are plotted against pH. A mechanism of Trolox C oxidation by LPO-II has been proposed in which protonation of an amino acid residue on LPO-II with a pKe of 2.3 is essential and ionization of the carboxylic acid group on Trolox C accelerates the reaction rate. The second-order rate constants were determined to be (4.1 +/- 0.5) x 10(6) M-1s-1 for protonated Trolox C oxidation and (1.9 +/- 0.3) x 10(7) M-1s-1 for deprotonated Trolox C.