Oxidative damage to mitochondrial complex I due to peroxynitrite: identification of reactive tyrosines by mass spectrometry.
There is growing evidence that oxidative phosphorylation (OXPHOS) generates reactive oxygen and nitrogen species within mitochondria as unwanted byproducts that can damage OXPHOS enzymes with subsequent enhancement of free radical production. The accumulation of this oxidative damage to mitochondria in brain is thought to lead to neuronal cell death resulting in neurodegeneration. The predominant reactive nitrogen species in mitochondria are nitric oxide and peroxynitrite. Here we show that peroxynitrite reacts with mitochondrial membranes from bovine heart to significantly inhibit the activities of complexes I, II, and V (50-80%) but with less effect upon complex IV and no significant inhibition of complex III. Because inhibition of complex I activity has been a reported feature of Parkinson's disease, we undertook a detailed analysis of peroxynitrite-induced modifications to proteins from an enriched complex I preparation. Immunological and mass spectrometric approaches coupled with two-dimensional PAGE have been used to show that peroxynitrite modification resulting in a 3-nitrotyrosine signature is predominantly associated with the complex I subunits, 49-kDa subunit (NDUFS2), TYKY (NDUFS8), B17.2 (17.2-kDa differentiation associated protein), B15 (NDUFB4), and B14 (NDUFA6). Nitration sites and estimates of modification yields were deduced from MS/MS fragmentograms and extracted ion chromatograms, respectively, for the last three of these subunits as well as for two co-purifying proteins, the beta and the d subunits of the F1F0-ATP synthase. Subunits B15 (NDUFB4) and B14 (NDUFA6) contained the highest degree of nitration. The most reactive site in subunit B14 was Tyr122, while the most reactive region in B15 contained 3 closely spaced tyrosines Tyr46, Tyr50, and Tyr51. In addition, a site of oxidation of tryptophan was detected in subunit B17.2 adding to the number of post-translationally modified tryptophans we have detected in complex I subunits (Taylor, S. W., Fahy, E., Murray, J., Capaldi, R. A., and Ghosh, S. S. (2003) J. Biol. Chem. 278, 19587-19590). These sites of oxidation and nitration may be useful biomarkers for assessing oxidative stress in neurodegenerative disorders.
Murray
James
J
Department of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Taylor
Steven W
SW
Zhang
Bing
B
Ghosh
Soumitra S
SS
Capaldi
Roderick A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019636:Neurodegenerative Diseases / Q000209:etiology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000633:toxicity*; D021122:Protein Subunits; D014443:Tyrosine / Q000378:metabolism*
D021122:Protein Subunits; D030421:Peroxynitrous Acid; D014443:Tyrosine; D042967:Electron Transport Complex I