An Electron Spin Resonance Spin-trapping Investigation of the Free Radicals Formed by the Reaction of Mitochondrial Cytochromec Oxidase with H2O2

Chen, Yeong‐Renn; Gunther, Michael R.; Mason, Ronald P.

doi:10.1074/jbc.274.6.3308

Cited by 72 publications

(73 citation statements)

References 46 publications

(29 reference statements)

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“…At higher concentrations of DMPO (up to 800 mM), the contribution of DMPO/ ⅐ ScysCcO relative to DMPO/ ⅐ CN was diminished, consistent with DMPO trapping the ⅐ CN as the primary radical and preventing the formation of ⅐ ScysCcO (data not shown). This DMPO/ ⅐ ScysCcO radical adduct was previously detected when H 2 O 2 was added to CcO (40) and did not contribute to the spectra shown in Figs. 1A or 2A obtained from the reaction mixture after a 1-h incubation.…”

Section: Dmpo Spin Trapping Of the Cyanyl Radical In The Reactionsupporting

confidence: 61%

“…Each spectrum shown in Fig. 3A was simulated using three species, DMPO/ ⅐ CN, DMPO/ ⅐ OH, and DMPO/ ⅐ ScysCcO protein-derived thiyl radical (a N ϭ 14.68, a H ␤ ϭ 15.74 G, and line width ϭ 3.12 G) (40). The formation of DMPO/ ⅐ ScysCcO was prevented by the pretreatment of CcO with N-ethylmaleimide.…”

Section: Dmpo Spin Trapping Of the Cyanyl Radical In The Reactionmentioning

confidence: 99%

“…1A. B, the spectra in A were simulated using three species: DMPO/ ⅐ OH, DMPO/ ⅐ CN, and DMPO/ ⅐ ScysCcO, a spin-trapped, protein-derived thiyl radical (40). The parameters of the computer simulation for DMPO/ ⅐ OH and DMPO/ ⅐ CN are the same as those described in Fig.…”

Section: Fig 3 Time Dependence Of Radical Adducts From the Reactionmentioning

confidence: 99%

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Electron Spin Resonance Investigation of the Cyanyl and Azidyl Radical Formation by Cytochrome c Oxidase

Chen

Sturgeon

Gunther

et al. 1999

Journal of Biological Chemistry

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Cyanide (CN؊ ) is a frequently used inhibitor of mitochondrial respiration due to its binding to the ferric heme a 3 of cytochrome c oxidase (CcO). As-isolated CcO oxidized cyanide to the cyanyl radical ( ⅐ CN) that was detected, using the ESR spin-trapping technique, as the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/ ⅐ CN radical adduct. The enzymatic conversion of cyanide to the cyanyl radical by CcO was time-dependent but not affected by azide (N 3 ؊ ). The small but variable amounts of compound P present in the as-isolated CcO accounted for this one-electron oxidation of cyanide to the cyanyl radical. In contrast, as-isolated CcO exhibited little ability to catalyze the oxidation of azide, presumably because of azide's lower affinity for the CcO. However, the DMPO/ ⅐ N 3 radical adduct was readily detected when H 2 O 2 was included in the system. The results presented here indicate the need to re-evaluate oxidative stress in mitochondria "chemical hypoxia" induced by cyanide or azide to account for the presence of highly reactive free radicals.Cyanide has been widely used as an inhibitor to study enzymatic hemoproteins such as oxidases and peroxidases (1). The unique ability of cyanide to inhibit aa 3 -type cytochrome oxidase is extremely important for investigating the enzyme complexes of the biological respiratory chain (2). Cyanide has long been known to serve as a ligand for the heme a 3 based on its ability to change the visible spectral characteristics of the heme environment (3, 4). Kinetic and mechanistic questions relating to the reaction of cytochrome c oxidase (CcO) 1 with cyanide have been the subject of many recent experiments (5-7). In addition to inhibiting hemoprotein-catalyzed reactions, cyanide is actively metabolized to carbon dioxide in biological systems (8). The cyanyl radical ( ⅐ CN) can be generated by one-electron oxidation of cyanide and detected with spin trapping in chemical (9) and electrochemical (10, 11) oxidizing systems. Previous studies (12, 13) indicated that peroxidases such as horseradish peroxidase, lactoperoxidase, or chloroperoxidase can use cyanide as a substrate and catalyze the one-electron oxidation to the cyanyl radical in the presence of H 2 O 2 . A similar result was also reported in the study of lignin peroxidase H2 from the white rot fungus Phanerochaete chrysosporium (14). Pharmacologically, cyanide is used as a chemical model of ischemia (15-19) because of its ability to block oxidative phosphorylation and thereby induce the production of reactive oxygen species. Cyanide-induced neurotoxicity and oxidative stress have also recently received much attention (20,21). It has been proposed that cyanide can activate the N-methyl-D-aspartate receptors for signal transduction (22, 23) and trigger the enzymatic pathways required for the generation of nitric oxide and reactive oxygen species, which are associated with lipid peroxidation in the nervous system.Azide also exhibits inhibitory effects on the mitochondrial CcO (2) and other hemoproteins such as catalase (24,25...

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Section: Dmpo Spin Trapping Of the Cyanyl Radical In The Reactionsupporting

confidence: 61%

Section: Dmpo Spin Trapping Of the Cyanyl Radical In The Reactionmentioning

confidence: 99%

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Electron Spin Resonance Investigation of the Cyanyl and Azidyl Radical Formation by Cytochrome c Oxidase

Chen

Sturgeon

Gunther

et al. 1999

Journal of Biological Chemistry

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“…However, protein radicals are now well recognized as normal intermediates for an increasing number of enzymes and some peroxidase-like hemoproteins. Examples of such radical intermediates include the thiyl radical of ribonucleotide reductase (29 -31); the tyrosyl radicals of prostaglandin H synthase (32), cytochrome c (33), and horse heart myoglobin (34); and the tyrosyl and thiyl radicals of cytochrome c peroxidase (35) and hemoglobin (36,37). GSH, a cysteinecontaining tripeptide, is ubiquitously distributed in nature and is the most abundant low molecular weight thiol in mammalian cells.…”

mentioning

confidence: 99%

“…In contrast, ESR spin trapping using DMPO as a spin trap detects the transient DMPO/protein radical adducts. Unfortunately, only a few protein radicals such as tyrosyl radicals of myoglobin (34) and thiyl radicals of human myoglobin (39,40), hemoglobin (36), and cytochrome c oxidase (35) have been trapped by DMPO and detected by ESR, and even in these cases, high concentrations of proteins had to be used. Therefore, in the detection of protein-derived radicals in biological systems, the sensitive immunochemical detection of protein radical-derived DMPO nitrone adducts is not only complementary to the ESR DMPO spintrapping technique, but a usually superior replacement (37).…”

mentioning

confidence: 99%

Protein Radical Formation during Lactoperoxidase-mediated Oxidation of the Suicide Substrate Glutathione

Guo

Detweiler

Mason

2004

Journal of Biological Chemistry

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A novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts has been developed. In this study, we employed this new approach, which combines the specificity of spin trapping and the sensitivity of antigen-antibody interactions, to investigate protein radical formation from lactoperoxidase (LPO). When LPO reacted with GSH in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct using enzyme-linked immunosorbent assay and Western blotting. The formation of this nitrone adduct depended on the concentrations of GSH, LPO, and DMPO as well as pH values, and GSH could not be replaced by H 2 O 2 . The level of this nitrone adduct was decreased significantly by azide, catalase, ascorbate, iodide, thiocyanate, phenol, or nitrite. However, its formation was unaffected by chemical modification of free cysteine, tyrosine, and tryptophan residues on LPO. ESR spectra showed that a glutathiyl radical was formed from the LPO/GSH/DMPO system, but no protein radical adduct could be detected by ESR. Its formation was decreased by azide, catalase, ascorbate, iodide, or thiocyanate, whereas phenol or nitrite increased it. GSH caused marked changes in the spectrum of compound II of LPO, indicating that GSH binds to the heme of compound II, whereas phenol or nitrite prevented these changes and reduced compound II back to the native enzyme. GSH also dose-dependently inhibited the peroxidase activity of LPO as determined by measuring 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation. Taken together, these results demonstrate that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO.

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CytochromecOxidase and Models

Halime

Karlin

2011

Copper‐Oxygen Chemistry

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An Electron Spin Resonance Spin-trapping Investigation of the Free Radicals Formed by the Reaction of Mitochondrial Cytochromec Oxidase with H2O2

Cited by 72 publications

References 46 publications

Electron Spin Resonance Investigation of the Cyanyl and Azidyl Radical Formation by Cytochrome c Oxidase

Electron Spin Resonance Investigation of the Cyanyl and Azidyl Radical Formation by Cytochrome c Oxidase

Protein Radical Formation during Lactoperoxidase-mediated Oxidation of the Suicide Substrate Glutathione

CytochromecOxidase and Models

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