Kinetic studies were performed with microperoxidase-8 (Fe(III)MP-8), the proteolytic breakdown product of horse heart cytochrome c containing an octapeptide linked to an iron protoporphyrin IX. Mn(III) was substituted for Fe(III) in Mn(III)MP-8. The mechanism of formation of the reactive metal-oxo and metal-hydroperoxo intermediates of M(III)MP-8 upon reaction of H(2)O(2) with Fe(III)MP-8 and Mn(III)MP-8 was investigated by rapid-scan stopped-flow spectroscopy and transient EPR. Two steps (k(obs1) and k(obs2)) were observed and analyzed for the reaction of hydrogen peroxide with both catalysts. The plots of k(obs1) as function of [H(2)O(2)] at pH 8.0 and pH 9.1 for Fe(III)MP-8, and at pH 10.2 and pH 10.9 for Mn(III)MP-8, exhibit saturation kinetics, which reveal the accumulation of an intermediate. Double reciprocal plots of 1/k(obs1) as function of 1/[H(2)O(2)] at different pH values reveal a competitive effect of protons in the oxidation of M(III)MP-8. This effect of protons is confirmed by the linear dependence of 1/k(obs1) on [H(+)] showing that k(obs1) increases with the pH. The UV-visible spectra of the intermediates formed at the end of the first step (k(obs1)) exhibit a spectrum characteristic of a high-valent metal-oxo intermediate for both catalysts. Transient EPR of Mn(III)MP-8 incubated with an excess of H(2)O(2), at pH 11.5, shows the detection of a free radical signal at g approximately equal to 2 and of a resonance at g approximately equal to 4 characteristic of a Mn(IV) (S = 3/2) species. On the basis of these results, the following mechanism is proposed: (i) M(III)MP-8-OH(2) is deprotonated to M(III)MP-8-OH in a rapid preequilibrium step, with a pK(a) = 9.2 +/- 0.9 for Fe(III)MP-8 and a pK(a) = 11.2 +/- 0.3 for Mn(III)MP-8; (ii) M(III)MP-8-OH reacts with H(2)O(2) to form Compound 0, M(III)MP8-OOH, with a second-order rate constant k(1) = (1.3 +/- 0.6) x 10(6) M(-1) x s(-1) for Fe(III)MP-8 and k(1) = (1.6 +/- 0.9) x 10(5) M(-1) x s(-1) for Mn(III)MP-8; (iii) this metal-hydroperoxo intermediate is subsequently converted to a high-valent metal-oxo species, M(IV)MP-8=O, with a free radical on the peptide (R(*+)). The first-order rate constants for the cleavage of the hydroperoxo group are k(2) = 165 +/- 8 s(-1) for Fe(III)MP-8 and k(2) = 145 +/- 7 s(-1) for Mn(III)MP-8; and (iv) the proposed M(IV)MP-8=O(R(*+)) intermediate slowly decays (k(obs2)) with a rate constant of k(obs2) = 13.1 +/- 1.1 s(-)(1) for Fe(III)MP-8 and k(obs2) = 5.2 +/- 1.2 s(-1) for Mn(III)MP-8. The results show that Compound 0 is formed prior to what is analyzed as a high-valent metal-oxo peptide radical intermediate.

This study describes the catalytic properties of manganese microperoxidase 8 [Mn(III)MP8] compared to iron microperoxidase 8 [Fe(III)MP8]. The mini-enzymes were tested for pH-dependent activity and operational stability in peroxidase-type conversions, using 2-methoxyphenol and 3,3'-dimethoxybenzidine, and in a cytochrome P450-like oxygen transfer reaction converting aniline to para-aminophenol. For the peroxidase type of conversions the Fe to Mn replacement resulted in a less than 10-fold decrease in the activity at optimal pH, whereas the aniline para-hydroxylation is reduced at least 30-fold. In addition it was observed that the peroxidase type of conversions are all fully blocked by ascorbate and that aniline para-hydroxylation by Fe(III)MP8 is increased by ascorbate whereas aniline para-hydroxylation by Mn(III)MP8 is inhibited by ascorbate. Altogether these results indicate that different types of reactive metal oxygen intermediates are involved in the various conversions. Compound I/II, scavenged by ascorbate, may be the reactive species responsible for the peroxidase reactions, the polymerization of aniline and (part of) the oxygen transfer to aniline in the absence of ascorbate. The para-hydroxylation of aniline by Fe(III)MP8, in the presence of ascorbate, must be mediated by another reactive iron-oxo species which could be the electrophilic metal(III) hydroperoxide anion of microperoxidase 8 [M(III)OOH MP8]. The lower oxidative potential of Mn, compared to Fe, may affect the reactivity of both compound I/II and the metal(III) hydroperoxide anion intermediate, explaining the differential effect of the Fe to Mn substitution on the pH-dependent behavior, the rate of catalysis and the operational stability of MP8.

The mechanism of microperoxidase-8 (MP-8) mediated O-and N-dealkylation was investigated. In the absence of ascorbate (peroxidase mode), many unidentified polymeric products are formed and the extent of substrate degradation correlates (r 0.94) with the calculated substrate ionization potential, reflecting the formation of radical intermediates.In the presence of ascorbate (P450 mode) formation of polymeric products is largely prevented but, surprisingly, dealkylation is not affected. In addition, aromatic hydroxylation and oxidative dehalogenation is observed. The results exclude a radical mechanism and indicate the involvement of a (hydro)peroxo-iron heme intermediate in P450-type of heteroatom dealkylation.

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Microperoxidase-8, Fe(III)MP-8, the heme octapeptide obtained by horse heart cytochrome c digestion, was studied in the presence of H(2)O(2). A modified form of the catalyst was isolated by HPLC and showed a UV/visible spectrum similar to that of Fe(III)MP-8. ESI-MS measurements revealed a 16 Da increase in molecular mass for the modified catalyst when compared to Fe(III)MP-8, suggesting the insertion of an oxygen atom. ESI-MS(2) fragmentation measurements point at oxygen incorporation on the His18 residue of the octapeptide of the modified catalyst. Comparison of the (1)H NMR chemical shifts of the methyl protons of the porphyrin ring of Fe(III)MP-8 and the modified catalyst shows a large shift for especially the 3-methyl and 5-methyl resonances, whereas the other (1)H NMR chemical shifts are almost unaffected. These observations can best be ascribed to a reorientation of the histidine axial ligand. The latter is suggested to be the consequence of an oxygen insertion, possibly on the imidazole ring of His18, thereby corroborating the data obtained by ESI-MS(2). (1)H NMR NOE difference measurements on Fe(III)MP-8 and on the modified catalyst supported the assignment of the H(delta)2 and H(epsilon)1 protons of the His18 imidazole ring. The ring amine proton H(delta)1 could not be detected in both forms of the catalyst. For Fe(III)MP-8 this absence of the H(delta)1 resonance can be ascribed to fast H/D exchange. For the modified catalyst the NMR data are not contradictory, with an oxygen insertion on position delta1 of the His18 imidazole ring with a fast H/D exchanging hydroxyl proton. Together these data converge in suggesting the H(2)O(2) modified catalyst bears a hydroxylated His18 axial ligand. The mechanism that could underlie Fe(III)MP-8 axial histidine hydroxylation is further discussed.