Acta Pharm. 49 (1999) 137-147

Kinetics and mechanism of primary radical formation and scavenge in biomimetic heme iron/hydroperoxide system

BLAZENKA JURIŠIC

Toxicological Chemistry, Department of Pharmaceutics, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacica 1, 10000 Zagreb, Croatia

The water-soluble N-acetylmicroperoxidase-8, Ac-MP-8, neither aggregates nor forms an micro-oxo dimer and is suitable as ferric heme model for study of peroxidases in biomimetic heme iron/hydroperoxide systems. The fast kinetics for the elementary steps of Ac-MP-8-catalyzed reaction with HOOH under transient-state and pre-steady-state conditions has been studied by stopped-flow spectrometry. The oxidation of Ac-MP-8 to Compound I analog in the presence of HOOH in aqueous solution at pH 7.0 and ambient temperature (20.0 +/- 0.2 °C) is a bimolecular reaction with determined rate constant of k₂ = 3.2 107 mol ^-1 L s^-1.
Kinetics of Ac-MP-8 disappearance was observed at 390 nm. The curve following the kinetic trace was calculated by a double-exponential fit trace and the average values of k₂' of (3.8 +/- 0.7) 103 mol^-1 L s^-1 for the faster reaction step and k₂ '' of (3.8 +/- 0.8) 102 mol^-1 L s^-1 for the slower step could be determined.
The second-order rate constants for oxygen transfer from HOOH to the iron(III) porphyrin were determined also by diammonium 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonate), ABTS, as a convenient radical trap for the resultant higher-valent iron-oxo porphyrin species. Reactions were monitored spectrometrically at 660 nm by following the appearance of the radical ABTS ^.+. Under concentration conditions where ([HOOH] << K_M and [ABTS] >> [Ac-MP-8]), concentration-time profile for ABTS⁺ formation was accurately represented by a general mono-exponential integrated equation with rate constants of about (1.4 +/- 0.4) 10³ mol^-1 L s^-1. These conditions enabled to delineate the nature of oxo-intermediates formed and to discriminate between the effects on the radical formation and on the radical scavenging, which are two major initial steps of mechanisms for an antioxidant action. The data are compared with those from earlier steady-state kinetic studies and demonstrate the importance of single turnover experiments.

Keywords: N-alfa-acetylmicroperoxidase-8, hydrogen peroxide, Compound I, free radicals, ABTS, stopped-flow