The peroxidasic activity of the haem octapeptide microperoxidase-8 (MP-8): the kinetic mechanism of the catalytic reduction of H2O2by MP-8 using 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate)(ABTS) as reducing substrate

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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.