The goal of this study was to explore the occurrence of nitrated proteins in mitochondria given that these organelles are endowed with a mitochondrial nitric oxide (NO.-) synthase and considering the important role that mitochondria have in energy metabolism. Our hypothesis is that nitration of proteins constitutes a posttranslational modification by which NO.- exhibits long-term effects above and beyond those bioregulatory ones mediated through the interaction with cytochrome c oxidase. Our studies are aimed at understanding the mechanisms underlying the nitration of proteins in mitochondria and the biological significance of such a process in the cellular milieu. On promoting a sustained NO.- production by mitochondria, we investigated various aspects of protein nitration. Among them, the localization of nitrated proteins in mitochondrial subfractions, the identification of nitrated proteins through proteomic approaches, the characterization of affected pathways, and depiction of a target sequence. The biological relevance was analyzed by considering the turnover of native and nitrated proteins. In this regard, mitochondrial dysfunction, ensuing nitrative stress, may be envisioned as the result of accumulation of nitrated proteins, resulting from an overproduction of endogenous NO.- (this study), a failure in the proteolytic system to catabolize modified proteins, or a combination of both. Finally, this study allows one to gain understanding on the mechanism and nitrating species underlying mitochondrial protein nitration.

An apparent discrepancy arises about the role of calcium on the rates of oxygen consumption by mitochondria: mitochondrial calcium increases the rate of oxygen consumption because of the activation of calcium-activated dehydrogenases, and by activating mitochondrial nitric oxide synthase (mtNOS), decreases the rates of oxygen consumption because nitric oxide is a competitive inhibitor of cytochrome oxidase. To this end, the rates of oxygen consumption and nitric oxide production were followed in isolated rat liver mitochondria in the presence of either L-Arg (to sustain a mtNOS activity) or N(G)-monomethyl-L-Arg (NMMA, a competitive inhibitor of mtNOS) under State 3 conditions. In the presence of NMMA, the rates of State 3 oxygen consumption exhibited a K(0.5) of 0.16 microM intramitochondrial free calcium, agreeing with those required for the activation of the Krebs cycle. By plotting the difference between the rates of oxygen consumption in State 3 with L-Arg and with NMMA at various calcium concentrations, a K(0.5) of 1.2 microM intramitochondrial free calcium was obtained, similar to the K(0.5) (0.9 microM) of the dependence of the rate of nitric oxide production on calcium concentrations. The activation of dehydrogenases, followed by the activation of mtNOS, would lead to the modulation of the Krebs cycle activity by the modulation of nitric oxide on the respiratory rates. This would ensue in changes in the NADH/NAD and ATP/ADP ratios, which would influence the rate of the cycle and the oxygen diffusion.

Nitric oxide is generated in vivo by nitric-oxide synthase (NOS) during the conversion of L-Arg to citrulline. Using a variety of biological systems and approaches emerging evidence has been accumulated for the occurrence of a mitochondrial NOS (mtNOS), identified as the alpha isoform of neuronal or NOS-1. Under physiological conditions, the production of nitric oxide by mitochondria has an important implication for the maintenance of the cellular metabolism, i.e. modulates the oxygen consumption of the organelles through the competitive (with oxygen) and reversible inhibition of cytochrome c oxidase. The transient inhibition suits the continuously changing energy and oxygen requirements of the tissue; it is a short-term regulation with profound pathophysiological consequences. This review describes the identification of mtNOS and the role of posttranslational modifications on mtNOS' activity and regulation.

Haynes V, Traaseth NJ, Elfering S, Fujisawa Y, Giulivi C. Nitration of specific tyrosines in FoF1 ATP synthase and activity loss in aging. Am J Physiol Endocrinol Metab 298: E978 -E987, 2010. First published February 16, 2010 doi:10.1152/ajpendo.00739.2009.-It has been reported that C-nitration of proteins occurs under nitrative/oxidative stress; however, its role in pathophysiological situations is not fully understood. In this study, we determined that nitration of Tyr 345 and Tyr 368 in the ␤-subunit of the mitochondrial FoF1-ATPase is a major target for nitrative stress in rat liver under in vivo conditions. The chemical characteristics of these Tyr make them suitable for a facilitated nitration (solvent accessibility, consensus sequence, and pKa). Moreover, ␤-subunit nitration increased significantly with the age of the rats (from 4 to 80 weeks old) and correlated with decreased ATP hydrolysis and synthesis rates. Although its affinity for ATP binding was unchanged, maximal ATPase activity decreased between young and old rats by a factor of two. These changes directly impacted the available ATP concentration in vivo, and it was expected that they would affect multiple cellular ATP-dependent processes. For instance, at least 50% of available [ATP] in the liver of older rats would have to be committed to sustain maximal Na ϩ -K ϩ -ATPase activity, whereas only 30% would be required for young rats. If this requirement was not fulfilled, the osmoregulation and Na ϩ -nutrient cotransport in liver of older rats would be compromised. On the basis of our studies, we propose that targeted nitration of the ␤-subunit is an early marker for nitrative stress and aging. adenosine 5=-triphosphate; adenosine 5=-triphosphatase; mitochondria; bioenergetics; biomarker IT HAS BEEN SHOWN THAT MITOCHONDRIA produce nitric oxide and that this molecule modulates the oxygen consumption of the organelle by competitive and noncompetitive inhibition of cytochrome c oxidase (18,30). Inhibition of oxygen consumption is transient as long as small quantities of nitric oxide are generated. However, at high or sustained levels of nitric oxide, the inhibition of the respiratory chain leads to an increased rate of formation of oxygen-free radicals that may damage mitochondria and/or initiate apoptosis (30). Other studies have demonstrated novel roles for nitric oxide through S-nitrosation of proteins (Ref. 43 and references therein). The finding that protein modification by nitrosation may be dynamically regulated and, in certain cases, coupled to cell surface signals has potential implications for other signaling pathways and cellular control mechanisms.However, in addition to its transient effects mediated through interaction with cytochrome c oxidase and/or S-nitrosation of proteins, nitric oxide may exhibit relatively long-term effects through the nitration of proteins (32,38). It has been proposed that C-nitration of proteins constitutes a novel chemical modification that occurs as a consequence of the "normal" oxidative background (39...