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

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 a(3)Cu(B) 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 100 mol 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 a(3)-Cu(B) binuclear centre was the initial reaction site. When the CcO was pre-treated with 10 mM (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

Endothelial NOS activity and myocardial oxygen metabolism define the salvageable ischemic time window for ischemic postconditioning

Ischemic postconditioning (IPOC) could be ineffective or even detrimental if the index ischemic duration is either too short or too long. The present study is to demonstrate that oxygen supply and metabolism defines a salvageable ischemic time window of IPOC in mice. C57BL/6 mice underwent coronary artery occlusion followed by reperfusion (I/R), with or without IPOC by three cycles of 10 s/10 s R/I. In vivo myocardial tissue oxygenation was monitored with electron paramagnetic resonance oximetry. Regional blood flow (RBF) was measured with a laser Doppler monitor. At the end of 60 min reperfusion, tissue from the risk area was collected, and mitochondrial enzyme activities were assayed. Tissue oximetry demonstrated that I/R induced a reperfusion hyperoxygenation state in the 30- and 45-min but not 15- and 60-min ischemia groups. IPOC attenuated the hyperoxygenation with 45 but not 30 min ischemia. RBF, eNOS phosphorylation, and mitochondrial enzyme activities were suppressed after I/R with different ischemic time, and IPOC afforded protection with 30 and 45 but not 60 min ischemia. Infarct size measurement indicated that IPOC reduced infarction with 30 and 45 min but not 60 min ischemia. Clearly, IPOC protected mouse heart with a defined ischemic time window between 30 and 45 min. This salvageable time window was accompanied by the improvement of RBF due to increased phosphorylated eNOS and the preservation of mitochondrial oxygen consumption due to conserved mitochondrial enzyme activities. Interestingly, this salvageable ischemic time window was mirrored by tissue hyperoxygenation status in the postischemic heart