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1 Department of Medicine, University of Florida College of Medicine, Gainesville, Florida, USA
2 Department of Medicine, University of Florida College of Medicine, Gainesville, Florida, USA; Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida, USA
* To whom correspondence should be addressed. E-mail: zhangjl{at}medicine.ufl.edu.
Persistent inhibition of cytochrome c oxidase, a terminal enzyme of the mitochondrial electron transport chain by excessive nitric oxide (NO) derived from inflammation, polluted air and tobacco smoke, contributes to enhanced oxidant production and programmed cell death or apoptosis of lung cells. We sought to determine whether the long-term exposure of pulmonary artery endothelial cells (PAEC) to pathophysiological concentrations of NO causes persistent inhibition of complex IV through redox modification of its key cysteine residues located in a putative NO-sensitive motif. Prolonged exposure of porcine PAEC to 1 mM NOC-18 (a slow-releasing NO donor, equivalent to 1-5 µM NO) results in a gradual and persistent inhibition of complex IV that is concomitant with a reduction in ratios of mitochondrial glutathione (GSH) and oxidized GSH (GSSG). Overexpression of thioredoxin in mitochondria of PAEC attenuated the NO-induced loss of complex IV activities, suggesting a redox regulation of complex IV activity. Sequence analysis of complex IV subunits revealed a novel putative NO-sensitive motif in subunit II (S2). There are only two cysteine residues in porcine complex IV S2, which are located in the putative motif. Immunoprecipitation/Western blot analysis and "biotin switch" assay demonstrated that exposure of PAEC to 1 mM NOC-18 increased S-nitrosylation of complex IV S2 by 200%. Site-directed mutagenesis of these two cysteines of complex IV S2 attenuated NO-increased nitrosylation of complex IV S2. These results demonstrate for the first time that NO nitrosylates active site cysteines of complex IV, which is associated with persistent inhibition of complex IV. NO inhibition of complex IV via nitrosylation of NO-sensitive cysteine residues can be a novel up-stream event in the NO-complex IV signaling for NO toxicity in lung endothelial cells.
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