Nitroxide radicals as research tools: Elucidating the kinetics and mechanisms of catalase-like and "suicide inactivation" of metmyoglobin.

BACKGROUND: Metmyoglobin (MbFe(III)) reaction with H(2)O(2) has been a subject of study over many years. H(2)O(2) alone promotes heme destruction frequently denoted "suicide inactivation," yet the mechanism underlying H(2)O(2) dismutation associated with MbFe(III) inactivation remains obscure. METHODS: MbFe(III) reaction with excess H(2)O(2) in the absence and presence of the nitroxide was studied at pH 5.3-8.1 and 25°C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H(2)O(2) depletion, O(2) evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy. RESULTS: The rates of both H(2)O(2) dismutation and heme inactivation processes depend on [MbFe(III)], [H(2)O(2)] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe(III) molecule catalyzes the dismutation of 50±10 H(2)O(2) molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFe(III) while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFe(IV)O by H(2)O(2) and by nitroxide, respectively. CONCLUSIONS: The nitroxide effects on H(2)O(2) dismutation catalyzed by MbFe(III) demonstrate that MbFe(IV)O reduction by H(2)O(2) is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I-H(2)O(2) adduct, which decomposes to a MbFe(IV)O and an inactivated heme at a ratio of 25:1. GENERAL SIGNIFICANCE: The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.

Mode of action of poly(vinylpyridine-N-oxide) in preventing silicosis: effective scavenging of carbonate anion radical.

Small particles of crystalline silicon dioxide (crystallites) are exceptionally toxic. Inhalation of quartz crystallites causes silicosis, a devastating lung disease afflicting miners, particularly coal and stone workers. Poly(vinylpyridine-N-oxide)s (PVPNOs) have been applied in the prevention and treatment of silicosis, but their mode of action has been obscure. Recently, the sites of inducible *NO synthase activation and of nitrotyrosine formation were associated anatomically with the pathological quartz particle-caused lesions in the lungs. It has been suggested that the *NO formed combines rapidly with O2*- to yield ONOO-, a potential mediator of lung injury following silica exposure. Here, we show that PVPNOs do not react with peroxynitrite but scavenge exceptionally rapidly CO3*- radicals, which are produced in the decomposition of ONOO- in bicarbonate solutions. The rate constant for the reaction of CO3*- with PVPNO was found to be independent of the type and size of PVPNO, i.e., k = (1.9 +/- 0.2) x 10(5) M(-1) s(-1) per monomer. In contrast, the rate constant for the reaction of CO3*- with the small molecule 4-methylpyridine N-oxide did not exceed 1 x 10(4) M(-1) s(-1). The underlying reason for the difference is that, in the dissolved polymeric PVPNOs, the electrostatic repulsion between the N-oxide zwitterions destabilizes them, increasing dramatically their pKa. The protonated N-oxides at physiological pH have abstractable hydrogen atoms and are expected to react rapidly with CO3*-, just as cyclic hydroxylamines do. It is also shown that PVPNO inhibits tyrosine nitration by peroxynitrite at pH 7.6 in the presence of excess of CO2 in a concentration-dependent manner. Hence, binding of PVPNO to the quartz particles and eliminating CO3*- could prevent the killing of macrophages, the associated release of macrophage-recruiting cytokines, and the amplification of the local concentration of *NO by the recruited macrophages. The latter causes necrosis of the macrophage-infiltrated lung tissue and, upon repair of the necrotic lesion, results in the growth of the dysfunctional fibrotic tissue, which is the hallmark of silicosis.

Direct determination of the Gibbs' energy of formation of peroxynitrous acid.

The kinetics of decomposition of peroxynitrous acid (ONOOH) was investigated in the presence of 0.1-0.75 M HClO(4) and at a constant ionic strength. The decay rate of ONOOH decreased in the presence of H(2)O(2), approaching a limiting value well below 75 mM H(2)O(2). It also decreased in the presence of relatively low [HNO(2)] but did not approach a lower limiting value, since ONOOH reacts directly with HNO(2). The latter reaction corresponds to a HNO(2)- and H(+)-catalyzed isomerization of ONOOH to nitrate, and its third-order rate constant was determined to be 520 +/- 30 M(-)(2) s(-)(1). The mechanism of formation of O(2)NOOH from ONOOH in the presence of H(2)O(2) was also scrutinized. The results demonstrated that in the presence of 0.1-0.75 M HClO(4) and 75 mM H(2)O(2) the formation of O(2)NOOH is insignificant. The most important finding in this work is the reversibility of the reaction ONOOH + H(2)O right harpoon over left harpoon HNO(2) + H(2)O(2), and its equilibrium constant was determined to be (7.5 +/- 0.4) x 10(-)(4) M. Using this value, the Gibbs' energy of formation of ONOOH was calculated to be 7.1 +/- 0.2 kcal/mol. This figure is in good agreement with the value determined previously from kinetic data using parameters for radicals formed during homolysis of peroxynitrite.