The Nature of the Intermediates in the Reactions of Fe(III)- and Mn(III)-Microperoxidase-8 with H₂O₂:  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.

Formation and Decay of Hydroperoxo-Ferric Heme Complex in Horseradish Peroxidase Studied by Cryoradiolysis

“…This spectrum is in very good agreement with the spectrum of the (Fe 3ϩ P-OOH Ϫ ) complex of heme oxygenase, 2 and with the kinetically resolved spectrum of the same intermediate obtained in stopped-flow studies of the reaction of Mb mutant H64Q with H 2 O 2 (24). This intermediate of the reaction of H 2 O 2 with heme enzymes is very unstable at ambient conditions and previously was characterized only via decomposition of transient absorption spectra in stopped-flow studies or via freeze-quench techniques (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)24). The same (Fe 3ϩ P-OOH Ϫ ) complex is firmly identified as an intermediate of the reductive activation of the bound dioxygen in heme proteins, as demonstrated by cryoradiolytic reduction studies (26,29,30,(32)(33)(34)(35) and quantum chemical calculations (10,11,23).…”

Structure and quantum chemical characterization of chloroperoxidase compound 0, a common reaction intermediate of diverse heme enzymes

“…Characterization of this intermediate is difficult, because it is unstable and short-lived (17). Compound 0 (Fe-OOH Ϫ ) was initially reported at low temperatures by stoppedflow experiments (18).…”

Oxidation catalysis by iron and manganese porphyrins within enzyme‐like cages

“…A limitation to the use of MPs as homogeneous catalysts is the presence of a degradative pathway, causing porphyrin bleaching during catalysis. However, they have been useful to well elucidate the effect of the intrinsic nature of the metal ion in modulating heme‐protein activity . Mn(III)‐MP8 is less efficient than Fe(III)‐MP8 in H 2 O 2 deprotonation, as revealed by the pH dependence of the kinetic constant for compound I intermediate formation (pK a 9.2 and 11.0 for iron and manganese, respectively) .…”