Electron-nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase: identification of the free radical site with a methionyl cluster.

Hoffman, Brian M.; Roberts, James E.; Brown, Theodore G.; Kang, Chae Hee; Margoliash, E.

doi:10.1073/pnas.76.12.6132

Cited by 65 publications

(36 citation statements)

References 23 publications

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“…33 While the intermediate product of cytochrome c peroxidase with substrate contains its two oxidizing equivalents in the form of a ferryl ion and and a free radical of an amino acid in the protein part of the molecule. 34 - 35 Recently, Lambeir et al 36 showed that PGH synthase reacts with organic hydroperoxides and fatty acid hydroperoxides in milliseconds to generate an intermediate which is spectrally similar to compound I of horseradish peroxidase. Compound I of PGH synthase is then converted to compound II within 170 msec.…”

Section: Resultsmentioning

confidence: 99%

PGH synthase and lipoxygenase generate superoxide in the presence of NADH or NADPH.

Kukreja¹,

Kontos²,

Heß³

et al. 1986

Circulation Research

458

210

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Purified PGH synthase when acting on arachidonic acid in the presence of reduced nicotinamideadenine dinucleotide or reduced nicotinamide-adenine dinucleotide 3'-phosphate generated superoxide in burst-like fashion. In eight experiments using different batches of enzyme, the mean ± SE rate of superoxide generation from 100 U of enzyme measured as the superoxide dismutase-inhibitable reduction of cytochrome c was 5.06 ± 0.19 nmol/min in the first minute and 0.35 ± 0.03 nmol/min subsequently. Optimum rates of superoxide were seen at concentrations of reduced nicotinamideadenine dinucleotide in excess of 80 /xM and reduced nicotinamide-adenine dinucleotide 3-phosphate in excess of 100 juM. Using prostaglandin G 2 or linoleic acid as substrate rather than arachidonate also resulted in superoxide generation. When prostaglandin H 2 was used as substrate, no superoxide was generated. The rate of superoxide generation was markedly inhibited by cyclooxygenase inhibitors. Superoxide generation was also observed during the action of lipoxygenase on either linoleic or arachidonic acid in the presence of reduced nicotinamide-adenine dinucleotide or reduced nicotinamide-adenine dinucleotide 3-phosphate but not in their absence. Indomethacin had no effect on superoxide generation from lipoxygenase. We conclude that PGH synthase and lipoxygenase produce superoxide via a side-chain reaction dependent on the presence of suitable reducing cosubstrate. This mechanism is analogous to that described for peroxidases in general. and topical application of arachidonate 89 cause a spectrum of functional, morphological, and biochemical cerebral arteriolar abnormalities. These consist of sustained dilation, reduced responsiveness to vasoconstrictor and vasodilator influences, focal destructive lesions of the endothelium and vascular smooth muscle, and reduced oxygen consumption of the vessel wall. Superoxide anion and other reactive agents derived from it, such as hydrogen peroxide and the hydroxyl radical, are the likely mediators of the cerebral arteriolar abnormalities seen in these conditions, since the latter are inhibited by scavengers of superoxide anion, hydrogen peroxide, and of the hydroxyl radical. 6 Received January 21, 1986; accepted August 25, 1986. ioles to these agents for more than a few minutes causes vascular injury. The enzymatic source of superoxide anion in the experimental conditions noted above is not firmly established. The following evidence suggests strongly that superoxide is produced via the action of the prostaglandin hydroperoxidase. First, the cerebral arteriolar abnormalities noted above can be reproduced by topical application of prostaglandin G 2 (PGG 2 ) 8 or 15-hydroperoxy-eicosatetraenoic acid (15-HPETE)," which are good substrates of the prostaglandin hydroperoxidase. The cerebral arteriolar abnormalities are not produced by prostaglandin H 2 (PGH 2 ), the product of the reaction catalyzed by prostaglandin hydroperoxidase.8 Second, the vasodilation and cerebral arteriolar abnormalities due to hype...

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Section: Resultsmentioning

confidence: 99%

PGH synthase and lipoxygenase generate superoxide in the presence of NADH or NADPH.

Kukreja¹,

Kontos²,

Heß³

et al. 1986

Circulation Research

458

210

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“…[11] At about the same time, ENDOR measurements on the CCP ES radical by Brian Hoffman and coworkers were interpreted in terms of a nucleophilically stabilized methionyl radical. [12] The advent of site-directed mutagenesis helped resolve some of the uncertainty surrounding the identity of the CCP ES radical. Dave Goodin, Grant Mauk, and Michael Smith demonstrated that Met 172 could not be the site of this radical, [13] and measurements from Kraut’s laboratory on the CCP Trp 51 Phe mutant demonstrated that this residue was not the source of the radical signal.…”

Section: Free Radicals In Proteinsmentioning

confidence: 99%

The Rise of Radicals in Bioinorganic Chemistry

Gray

2016

Israel Journal of Chemistry

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Prior to 1950, the consensus was that biological transformations occurred in two-electron steps, thereby avoiding the generation of free radicals. Dramatic advances in spectroscopy, biochemistry, and molecular biology have led to the realization that protein-based radicals participate in a vast array of vital biological mechanisms. Redox processes involving high-potential intermediates formed in reactions with O2 are particularly susceptible to radical formation. Clusters of tyrosine (Tyr) and tryptophan (Trp) residues have been found in many O2-reactive enzymes, raising the possibility that they play an antioxidant protective role. In blue copper proteins with plastocyanin-like domains, Tyr/Trp clusters are uncommon in the low-potential single-domain electron-transfer proteins and in the two-domain copper nitrite reductases. The two-domain muticopper oxidases, however, exhibit clusters of Tyr and Trp residues near the trinuclear copper active site where O2 is reduced. These clusters may play a protective role to ensure that reactive oxygen species are not liberated during O2 reduction.

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“…In contrast, reaction of CCP with hydrogen peroxide produces an oxidized intermediate, compound ES, that is analogous to compound I except that the porphyrin radical cation is absent. Instead, an electron paramagnetic resonance (EPR) signal is observed at g = 2 that is only weakly magnetically coupled to the (Fe4+=O) iron center and this signal has generally been attributed to a free radical residing on a reversibly oxidized protein residue (10)(11)(12). As compound ES is believed to be an intermediate in the catalytic oxidation of ferrocytochrome c by CCP, detailed understanding of this form of the enzyme is of considerable importance.…”

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confidence: 99%

“…In addition, Trp-51 is replaced by phenylalanine, a less easily oxidized residue, in all other peroxidases and catalase (2). However, Hoffman and coworkers (12,16) have performed an extensive study of the EPR and electron nuclear double resonance (ENDOR) properties of the radical species at liquid helium temperature and have concluded that the data are completely inconsistent with the existence of either a neutral or cationic tryptophan radical. Instead, they proposed that a nucleophilically stabilized sulfur radical was consistent with the intrinsic anisotropy of the EPR signal at 4 K. The most likely candidate for such a center is Met-172, which lies 3.7 A below the proximal heme face (3).…”

mentioning

confidence: 99%

Studies of the radical species in compound ES of cytochrome c peroxidase altered by site-directed mutagenesis.

Goodin¹,

Mauk²,

Smith³

1986

Proc. Natl. Acad. Sci. U.S.A.

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Yeast cytochrome c peroxidase reacts with hydrogen peroxide to form an intermediate, compound ES, in which the heme iron atom is converted to a ferryl function (Fe4" =O) and a radical center is generated on a reversibly oxidizable amino acid residue of uncertain identity. As methionine-172 is a possible site of this radical, we have constructed specific variants of cytochrome c peroxidase in which methionine-172 is replaced by serine or cysteine. These mutants and the wild-type enzyme have been expressed in Saccharomyces cerevisiae, purified, and crystallized. Both mutant enzymes are fully active. A stable, reversible, peroxide-induced intermediate with optical properties characteristic of compound ES is observed for the three forms of the enzyme. The electron paramagnetic resonance spectrum of this intermediate at 93 K for the serine mutant exhibits the narrow free-radical signal and hyperfine structure observed for the wild-type enzyme. However, a broader component of the signal that is observed for the wild-type enzyme at this temperature is absent from the spectrum observed with the serine mutant. These results demonstrate that the narrow component of the free-radical signal observed at 93 K cannot reside at methionine-172. The absence of the broader component of the signal for the serine mutant may reflect the loss of spin density on methionine or, alternatively, could arise from conformationally induced changes in the properties of the radical. The results are consistent with a heterogeneity of radical species in the ES complex.

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Electron-nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase: identification of the free radical site with a methionyl cluster.

Cited by 65 publications

References 23 publications

PGH synthase and lipoxygenase generate superoxide in the presence of NADH or NADPH.

PGH synthase and lipoxygenase generate superoxide in the presence of NADH or NADPH.

The Rise of Radicals in Bioinorganic Chemistry

Studies of the radical species in compound ES of cytochrome c peroxidase altered by site-directed mutagenesis.

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