title: The nature of the intermediates in the reactions of Fe(III)- and Mn(III)-microperoxidase-8 with H(2)O(2): a rapid kinetics study.
abstract: 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.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Primus
authors.forename: Sylvie
authors.initials: S
authors.lastname: Grunenwald
authors.forename: Peter-Leon
authors.initials: PL
authors.lastname: Hagedoorn
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Albrecht-Gary
authors.forename: Dominique
authors.initials: D
authors.lastname: Mandon
authors.forename: Cees
authors.initials: C
authors.lastname: Veeger
mesh_terms: D000818:Animals; D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005290:Ferric Compounds; D008345:Manganese; D006861:Hydrogen Peroxide; D010544:Peroxidases; C021799:microperoxidase