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 H2O2 with Fe(III)MP-8 and Mn(III)MP-8 was investigated by rapid-scan stopped-flow spectroscopy and transient EPR. Two steps (kobs1 and kobs2) were observed and analyzed for the reaction of hydrogen peroxide with both catalysts. The plots of kobs1 as function of [H2O2] 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/kobs1 as function of 1/[H2O2] 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/kobs1 on [H+] showing that kobs1 increases with the pH. The UV-visible spectra of the intermediates formed at the end of the first step (kobs1) 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 H2O2, at pH 11.5, shows the detection of a free radical signal at g ≈ 2 and of a resonance at g ≈ 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-OH2 is deprotonated to M(III)MP-8-OH in a rapid preequilibrium step, with a pKa = 9.2 ± 0.9 for Fe(III)MP-8 and a pKa = 11.2 ± 0.3 for Mn(III)-MP-8; (ii) M(III)MP-8-OH reacts with H2O2 to form Compound 0, M(III)MP8-OOH, with a second-order rate constant k1 = (1.3 ± 0.6) × 106 M-1·s-1 for Fe(III)MP-8 and k1 = (1.6 ± 0.9) × 105 M-1·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 k2 = 165 ± 8 s-1 for Fe(III)MP-8 and k2 = 145 ± 7 s-1 for Mn(III)MP-8; and (iv) the proposed M(IV)MP-8=O(R·+) intermediate slowly decays (kobs2) with a rate constant of kobs2 = 13.1 ± 1.1 s-1 for Fe(III)MP-8 and kobs2 = 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.