Electron-paramagnetic-resonance studies on a photochemically produced species of horseradish peroxidase compound I.
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ABSTRACT:
SUBMITTER: McIntosh AR
PROVIDER: S-EPMC1183618 | biostudies-other | 1977 Oct
REPOSITORIES: biostudies-other
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Project description:The fleeting ferric peroxo and hydroperoxo intermediates of dioxygen activation by hemoproteins can be readily trapped and characterized during cryoradiolytic reduction of ferrous hemoprotein-O2 complexes at 77 K. Previous cryoannealing studies suggested that the relaxation of cryogenerated hydroperoxoferric intermediates of myoglobin (Mb), hemoglobin, and horseradish peroxidase (HRP), either trapped directly at 77 K or generated by cryoannealing of a trapped peroxo-ferric state, proceeds through dissociation of bound H2O2 and formation of the ferric heme without formation of the ferryl porphyrin π-cation radical intermediate, compound I (Cpd I). Herein we have reinvestigated the mechanism of decays of the cryogenerated hydroperoxyferric intermediates of α- and β-chains of human hemoglobin, HRP, and chloroperoxidase (CPO). The latter two proteins are well-known to form spectroscopically detectable quasistable Cpds I. Peroxoferric intermediates are trapped during 77 K cryoreduction of oxy Mb, α-chains, and β-chains of human hemoglobin and CPO. They convert into hydroperoxoferric intermediates during annealing at temperatures above 160 K. The hydroperoxoferric intermediate of HRP is trapped directly at 77 K. All studied hydroperoxoferric intermediates decay with measurable rates at temperatures above 170 K with appreciable solvent kinetic isotope effects. The hydroperoxoferric intermediate of β-chains converts to the S = 3/2 Cpd I, which in turn decays to an electron paramagnetic resonance (EPR)-silent product at temperature above 220 K. For all the other hemoproteins studied, cryoannealing of the hydroperoxo intermediate directly yields an EPR-silent majority product. In each case, a second follow-up 77 K γ-irradiation of the annealed samples yields low-spin EPR signals characteristic of cryoreduced ferrylheme (compound II, Cpd II). This indicates that in general the hydroperoxoferric intermediates relax to Cpd I during cryoanealing at low temperatures, but when this state is not captured by reaction with a bound substrate, it is reduced to Cpd II by redox-active products of radiolysis.
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Project description:Polychlorinated biphenyls (PCBs) can be oxygenated to form very reactive hydroquinone and quinone products. A guiding hypothesis in the PCB research community is that some of the detrimental health effects of some PCBs are a consequence of these oxygenated forms undergoing one-electron oxidation or reduction, generating semiquinone radicals (SQ (*-)). These radicals can enter into a futile redox cycle resulting in the formation of reactive oxygen species, that is, superoxide and hydrogen peroxide. Here, we examine some of the properties and chemistry of these semiquinone free radicals. Using electron paramagnetic resonance (EPR) to detect SQ (*-) formation, we observed that (i) xanthine oxidase can reduce quinone PCBs to the corresponding SQ (*-); (ii) the heme-containing peroxidases (horseradish and lactoperoxidase) can oxidize hydroquinone PCBs to the corresponding SQ (*-); (iii) tyrosinase acting on PCB ortho-hydroquinones leads to the formation of SQ (*-); (iv) mixtures of PCB quinone and hydroquinone form SQ (*-) via a comproportionation reaction; (v) SQ (*-) are formed when hydroquinone-PCBs undergo autoxidation in high pH buffer (approximately >pH 8); and, surprisingly, (vi) quinone-PCBs in high pH buffer can also form SQ (*-); (vii) these observations along with EPR suggest that hydroxide anion can add to the quinone ring; (viii) H 2 O 2 in basic solution reacts rapidly with PCB-quinones; and (ix) at near-neutral pH SOD can catalyze the oxidization of PCB-hydroquinone to quinone, yielding H 2 O 2. However, using 5,5-dimethylpyrroline-1-oxide (DMPO) as a spin-trapping agent, we did not trap superoxide, indicating that generation of superoxide from SQ (*-) is not kinetically favorable. These observations demonstrate multiple routes for the formation of SQ (*-) from PCB-quinones and hydroquinones. Our data also point to futile redox cycling as being one mechanism by which oxygenated PCBs can lead to the formation of reactive oxygen species, but this is most efficient in the presence of SOD.
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Project description:Tryptophan-based free radicals have been implicated in a myriad of catalytic and electron transfer reactions in biology. However, very few of them have been trapped so that biophysical characterizations can be performed in a high-precision context. In this work, tryptophan derivative-based radicals were studied by high-frequency/high-field electron paramagnetic resonance (HFEPR) and quantum chemical calculations. Radicals were generated at liquid nitrogen temperature with a photocatalyst, sacrificial oxidant, and violet laser. The precise g-anisotropies of l- and d-tryptophan, 5-hydroxytryptophan, 5-methoxytryptophan, 5-fluorotryptophan, and 7-hydroxytryptophan were measured directly by HFEPR. Quantum chemical calculations were conducted to predict both neutral and cationic radical spectra for comparison with the experimental data. The results indicate that under the experimental conditions, all radicals formed were cationic. Spin densities of the radicals were also calculated. The various line patterns and g-anisotropies observed by HFEPR can be understood in terms of spin-density populations and the positioning of oxygen atom substitution on the tryptophan ring. The results are considered in the light of the tryptophan and 7-hydroxytryptophan diradical found in the biosynthesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase.
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