Heme-(hydro)peroxide catalysed O- and N-dealkylation : a study with microperoxidase

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

Isolation and characterization of a microperoxidase-8 with a modified histidine axial ligand

Microperoxidase-8, Fe(III)MP-8, the heme octapeptide obtained by horse heart cytochrome c digestion, was studied in the presence of H 2O 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 1H 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 1H 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. 1H NMR NOE difference measurements on Fe(III)MP-8 and on the modified catalyst supported the assignment of the Hd2 and He1 protons of the His18 imidazole ring. The ring amine proton Hd1 could not be detected in both forms of the catalyst. For Fe(III)MP-8 this absence of the Hd1 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 d1 of the His18 imidazole ring with a fast H/D exchanging hydroxyl proton. Together these data converge in suggesting the H 2O 2 modified catalyst bears a hydroxylated His18 axial ligand. The mechanism that could underlie Fe(III)MP-8 axial histidine hydroxylation is further discussed

The Nature of the intermediates in the reactions of Fe(III)- and Mn(III)-microperoxidase-8 with H2O2 : a rapid kinetic study

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) x 106 M-1?s-1 for Fe(III)MP-8 and k1 = (1.6 ± 0.9) x 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 intermediat

The effect of iron to manganese substitution on microperoxidase-8 catalysed peroxidase and cytochrome P450 type of catalysis

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

Reversible formation of high-valent-iron-oxo-porphyrin intermediate in heme-based catalysis: revisiting the kinetic model for horseradish peroxidase.

Many heme-containing biocatalysts exert their catalytic action through the initial formation of so-called high-valent-iron-oxo porphyrin intermediates. For horseradish peroxidase the initial intermediate formed has been identified as a high-valent-iron-oxo porphyrin π-radical cation, called compound I. A strongly held concept in the field of peroxidase-type of catalysis is the irreversible character of the reaction leading to formation of this compound I. Results of the present paper, however, point to reversibility of formation of the high-valent-iron-oxo porphyrin intermediate for various heme-containing catalysts, including horseradish peroxidase. This results in heme-catalysed exchange of the oxygens of H2O2 with those of H2O. The existence of this heme-catalysed oxygen exchange follows from the observation that upon incubation of 18O-labelled H182O2 with heme-containing biocatalysts significant loss of the 18O label from the H182O2, accompanied by the formation of unlabelled H2O2, is observed. Thus, for the heme biocatalysts studied, exchange of the oxygen of their high-valent-iron-oxo intermediate with that of water occurs rapidly. This observation implies the need for an update of the kinetic model for horseradish peroxidase. Revaluation and extension of the previous kinetic model showed the necessity to include several additional reaction steps, taking both reversible compound I formation and formation of enzyme-substrate complexes into account

Warping a single Mn acceptor wavefunction by straining the GaAs host

Transition-metal dopants such as Mn determine the ferromagnetism in dilute magnetic semiconductors such as Ga1-xMnxAs. Recently, the acceptor states of Mn dopants in GaAs were found to be highly anisotropic owing to the symmetry of the host crystal. Here, we show how the shape of such a state can be modified by local strain. The Mn acceptors near InAs quantum dots are mapped at room temperature by scanning tunnelling microscopy. Dramatic distortions and a reduction in the symmetry of the wavefunction of the hole bound to the Mn acceptor are observed originating from strain induced by quantum dots. Calculations of the acceptor-state wavefunction in the presence of strain, within a tight-binding model and within an effective-mass model, agree with the experimentally observed shape. The magnetic easy axes of strained lightly doped Ga1-xMnxAs can be explained on the basis of the observed local density of states for the single Mn spi

WELFARE REFORM: Recent Policy and Politics

Public policy research has not combined policy and political analysis as its pioneers imagined. Mostly, political scientists study just the policy process, abandoning prescriptions to policy specialists. A better approach is to combine policy and political analysis, using positions on issues to assess policymaking, and vice versa. This article illustrates that approach by applying it to recent welfare reform. I take raising work levels as the chief goal of reform and assess the Family Support Act (FSA) of 1988 and the Personal Responsibility and Work Opportunity Reconciliation Act (PRWORA) of 1996 against it. FSA was a timid compromise that achieved little while PRWORA was a conflicted measure that achieved more, but at unnecessary risk. I also ask how the tensions between best policy and politics might be resolved. This approach makes public policy research more relevant to the real stakes in policymaking. [142 words.] Copyright 2002 by The Policy Studies Organization.