The peroxidasic activity of the haem octapeptide microperoxidase-8 (MP-8): the kinetic mechanism of the catalytic reduction of H2O2 by MP-8 using 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate)(ABTS) as reducing substrate

Abstract

Kinetic studies of the MP-8-catalysed reduction of H₂O₂ using the chromophoric reducing substrate, ABTS, are reported. Over a wide range of MP-8, H₂O₂, and ABTS concentrations—under the con-ditions [ABTS] > 10 [H₂O₂] > 10 [MP-8]—the formation of the ABTS ⁺˙ cation radical adheres precisely to a first-order kinetic rate law over at least 90% reaction. Kinetic studies using cyclical variation of the concentration of each reactant under conditions where the concentrations of the other two were maintained constant revealed that the pseudo-first-order rate constant is a sum of three independent terms, implying three parallel kinetic processes. These processes are suggested to be:

(i) a ‘normal’ peroxidase-type catalytic cycle with free MP-8 as the catalyst, proceeding via hypervalent oxo-iron(IV) porphyrin intermediates, analogous to compounds I and II of the peroxidase enzymes;

(ii) a parallel cycle to (i) in which the catalyst is an MP-8· ABTS complex, as opposed to the free MP-8 of (i); and

(iii) a degradative process involving direct attack of H₂O₂ on the MP-8 porphyrin moiety. This degradative pathway is inhibited by ABTS in a manner which indicates that only MP-8, not the MP-8·ABTS complex, is susceptible to attack by H₂O₂.

The relevant kinetic parameter for the mechanistic study of iron-porphyrin peroxidasic model systems using ABTS is thus suggested to be the limiting first-order rate constant as both [ABTS] and [H₂O₂] tend to zero.