The Nature of the Intermediates in the Reactions of Fe(III)- and Mn(III)-Microperoxidase-8 with H2O2:  a Rapid Kinetics Study

• Published in: Journal of the American Chemical Society 2002-02, Vol.124 (7), p.1214-1221
• Main Authors: Primus, Jean-Louis, Grunenwald, Sylvie, Hagedoorn, Peter-Leon, Albrecht-Gary, Anne-Marie, Mandon, Dominique, Veeger, Cees
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja016907u

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 (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 [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/k obs1 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/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 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 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 H2O2 to form Compound 0, M(III)MP8-OOH, with a second-order rate constant k 1 = (1.3 ± 0.6) × 106 M-1·s-1 for Fe(III)MP-8 and k 1 = (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 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 ra