scholarly journals Mechanism in the reaction of cytochrome c oxidase with organic hydroperoxides: an ESR spin-trapping investigation

2002 ◽  
Vol 365 (2) ◽  
pp. 461-469 ◽  
Author(s):  
Yeong-Renn CHEN ◽  
Ronald P. MASON
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Spin Trap ◽  
Spin Trapping ◽  
Initial Reaction ◽  
Radical Species ◽  
Alkoxyl Radical ◽  
Tyrosine Residues ◽  
Esr Spin Trapping ◽  
Organic Hydroperoxides

Organic hydroperoxides are of great utility in probing the reaction mechanism and the toxicological consequences of lipid peroxidation. In the present study, ESR spin-trapping was employed to investigate the peroxidation of mitochondrial cytochrome c oxidase (CcO) with t-butyl hydroperoxide (t-BuOOH) and cumene hydroperoxide (CumOOH). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to detect the radical species formed from the reaction of CcO with t-BuOOH. The presence of t-BuOOH-derived alkoxyl radical (t-BuO˙) as the primary radical indicates reductive scission of the O—O bond by CcO. The ESR signal of DMPO/˙Ot-Bu can be partially abolished by cyanide, implying that the reductive cleavage involved the haem a3CuB binuclear site of CcO. A nitroso spin trap, 2-methyl-2-nitrosopropane (MNP), was used to detect and identify radical species from the reaction of CcO with CumOOH. In addition to the t-BuOOH-derived methyl, hydroxylmethyl and tertiary carbon-centred radicals, a protein-derived radical was detected. The intensity of the ESR signal from the protein radical increased with the CumOOH concentration at low CumOOH/CcO ratios, with maximal intensity at a ratio of 100mol of CumOOH/mol of CcO. The immobilized protein radical adduct of MNP was stable and persistent after dialysis; it was also resistant to proteolytic digestion, suggesting that it was formed in the transmembrane region, a region that is not accessible to proteases. Its signal was greatly enhanced when CcO cysteine residues were chemically modified by N-ethylmaleimide, when the tryptophan residues in CcO were oxidized by N-bromosuccimide, and when tyrosine residues on the surface of CcO were iodinated, showing that a radical equilibrium was established among the cysteine, tryptophan and tyrosine residues of the protein-centred radical. Pre-treatment of CcO with cyanide prevented detectable MNP adduct formation, confirming that the haem a3-CuB binuclear centre was the initial reaction site. When the CcO was pre-treated with 10mM (100 equivalents) of CumOOH, the enzyme activity decreased by more than 20%. This inhibition was persistent after dialysis, suggesting that the detected protein-centred radical was, in part, involved in the irreversible inactivation by CumOOH. Visible spectroscopic analysis revealed that the haem a of CcO was not affected during the reaction. However, the addition of pyridine to the reaction mixture under alkaline conditions resulted in the destruction of the haem centre of CcO, suggesting that its protein matrix rather than its haem a is the target of oxidative damage by the organic hydroperoxide.

scholarly journals Formation of peroxide- and globin-derived radicals from the reaction of methaemoglobin and metmyoglobin with t-butyl hydroperoxide: an ESR spin-trapping investigation

1997 ◽  
Vol 322 (2) ◽  
pp. 633-639 ◽  
Author(s):  
Jolanda Van der ZEE
Keyword(s):  
Spin Trap ◽  
Hydrogen Abstraction ◽  
Spin Trapping ◽  
Tyrosyl Radical ◽  
Alkoxyl Radical ◽  
Butyl Hydroperoxide ◽  
Heterolytic Cleavage ◽  
Esr Spin Trapping ◽  
Radical Adduct ◽  
Protein Radicals

The reaction of human methaemoglobin and horse metmyoglobin with t-butyl hydroperoxide (t-BuOOH) was investigated with the ESR spin-trapping technique. With the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) the formation of peroxyl, alkoxyl and methyl radicals derived from t-BuOOH could be detected. The relative contributions of these radicals were determined at various DMPO concentrations by computer simulation. From these data it could be concluded that the alkoxyl radical was the initial radical produced, which indicates that the hydroperoxide is cleaved homolytically. Further investigations, with the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), showed the formation of globin-centred radicals. Non-specific proteolysis of the MNP adducts revealed isotropic three-line spectra, which means that the radical adducts were centred on a tertiary carbon with no bonds to a hydrogen or nitrogen. Comparison with MNP adducts of several amino acids indicated that in methaemoglobin the radical adduct was most probably located on a valine residue. With metmyoglobin the same adduct was obtained, whereas an additional adduct could be assigned to a tyrosyl radical. These protein radicals most probably resulted from hydrogen abstraction by the metal–oxo species, formed by heterolytic cleavage of the hydroperoxide. These results therefore show that homolytic cleavage of the hydroperoxide leads to the formation of peroxide-derived radicals, whereas concurrent heterolytic cleavage results in protein-derived radicals.

scholarly journals Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c

1975 ◽  
Vol 147 (1) ◽  
pp. 145-153 ◽  
Author(s):  
M T Wilson ◽  
C Greenwood ◽  
M Brunori ◽  
E Antonini
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Time Course ◽  
Kinetic Studies ◽  
Initial Reaction ◽  
Fast Phase ◽  
Ferrocytochrome C ◽  
Cytochrome C2 ◽  
Kinetics Of ◽  
Flow Experiments

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with ‘visible’ copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the ‘visible’ copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and ‘visible’ copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.

scholarly journals Spin-trapping studies on the free-radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions isolated hepatocytes and in vivo in the rat

1982 ◽  
Vol 204 (2) ◽  
pp. 593-603 ◽  
Author(s):  
E Albano ◽  
K A K Lott ◽  
T F Slater ◽  
A Stier ◽  
M C R Symons ◽  
...  
Keyword(s):  
Free Radical ◽  
Carbon Tetrachloride ◽  
Spin Trap ◽  
Peroxy Radical ◽  
Metabolic Activation ◽  
Spin Trapping ◽  
Metabolic Inhibitors ◽  
Radical Species ◽  
Free Radical Species ◽  
Trichloromethyl Radical

1. The metabolic activation of carbon tetrachloride to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury produced by this toxic agent. Electron-spin-resonance (e.s.r.) spin-trapping techniques were used to characterize the free-radical species involved. 2. Spin trapping was applied to the activation of carbon tetrachloride by liver microsomal fractions in the presence of NADPH, and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide (‘phenyl t-butyl nitrone’) (PBN) and [13C]carbon tetrachloride provide unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. 3. With the spin trap 2-methyl-2-nitrosopropane, however, the major free-radical species trapped are unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. 4. Although pulse radiolysis and other evidence support the very rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and oxygen, no clear evidence for the trapping of the peroxy radical was obtainable. 5. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. 6. High concentrations of the spin traps used were found to have significant effects on cytochrome P-450-mediated reactions; this requires caution in interpreting results of experiments done in the presence of PBN at concentrations greater than 50 mM.

scholarly journals Identification of protein-derived tyrosyl radical in the reaction of cytochrome c and hydrogen peroxide: characterization by ESR spin-trapping, HPLC and MS

2002 ◽  
Vol 363 (2) ◽  
pp. 281 ◽  
Author(s):  
Steven Yue QIAN ◽  
Yeong-Renn CHEN ◽  
Leesa J. DETERDING ◽  
Yang C. FANN ◽  
Colin F. CHIGNELL ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cytochrome C ◽  
Spin Trapping ◽  
Tyrosyl Radical ◽  
Esr Spin Trapping

scholarly journals The iron(III)-adriamycin complex inhibits cytochrome c oxidase before its inactivation

1988 ◽  
Vol 250 (3) ◽  
pp. 827-834 ◽  
Author(s):  
B B Hasinoff ◽  
J P Davey
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Kinetic Studies ◽  
Slow Inactivation ◽  
Radical Species ◽  
Inhibitor Complex ◽  
Antitumour Drug ◽  
Strong Contrast

Cytochrome c oxidase was found to be competitively inhibited by a complex formed between Fe3+ and the cardiotoxic antitumour drug adriamycin (doxorubicin) with an inhibition constant, Ki, of 12 microM. This competitive inhibition precedes the slower Fe3+-adriamycin induced inactivation of cytochrome c oxidase. In strong contrast with this result, free adriamycin was not observed to either inhibit or inactivate cytochrome c oxidase (Ki greater than 3 mM). Since, typically, polycations are known to inhibit cytochrome c oxidase, the competitive inhibition displayed by the Fe3+-adriamycin complex may also result from its polycationic character. Cytochrome c oxidase was also inhibited by pentan-1-ol (Ki 13 mM), and kinetic studies carried out in the presence of both inhibitors demonstrated that the Fe3+-adriamycin complex and pentan-1-ol are mutually exclusive inhibitors of cytochrome c oxidase. The inhibitor pentan-1-ol was also effective in preventing the slow inactivation of cytochrome c oxidase induced by Fe3+-adriamycin, presumably by blocking its binding to the enzyme. It is postulated that the slow inactivation of cytochrome c oxidase occurs when reactive radical species are produced while the Fe3+-adriamycin is complexed to cytochrome c oxidase in an enzyme-inhibitor complex. The Fe3+-adriamycin-induced inactivation of cytochrome c oxidase may be, in part, responsible for the cardiotoxicity of adriamycin.

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