Peroxynitrite Mediates Active Site Tyrosine Nitration in Manganese Superoxide Dismutase. Evidence of a Role for the Carbonate Radical Anion

Abstract

Protein tyrosine nitration has been observed in a variety of human diseases associated with oxidative stress, such as inflammatory, neurodegenerative and cardiovascular conditions. However, the pathways leading to nitration of tyrosine residues are still unclear. Recent studies have shown that peroxynitrite (PN), produced by the reaction of superoxide and nitric oxide, can lead to protein nitration and inactivation. Tyrosine nitration may also be mediated by nitrogen dioxide produced by the oxidation of nitrite by peroxidases. Manganese superoxide dismutase (MnSOD), which plays a critical role in cellular defense against oxidative stress by decomposing superoxide within mitochondria, is nitrated and inactivated under pathological conditions. In this study, MnSOD is shown to catalyze PN-mediated self-nitration. Direct, spectroscopic observation of the kinetics of PN decay and nitrotyrosine formation (kcat = 9.3 × 102 M-1s-1) indicates that the mechanism involves redox cycling between Mn2+ and Mn3+, similar to that observed with superoxide. Distinctive patterns of tyrosine nitration within MnSOD by various reagents were revealed and quantified by MS/MS analysis of MnSOD trypsin digest peptides. These analyses showed that three of the seven tyrosine residues of MnSOD (Tyr34, Tyr9, and Tyr11) were most susceptible to nitration and that the relative amounts of nitration of these residues varied widely depending upon the nature of the nitrating agent. Notably, nitration mediated by PN, both in the presence and absence of CO2, resulted in nitration of the active site tyrosine, Tyr34, while nitration by freely diffusing nitrogen dioxide led to surface nitration at Tyr9 and Tyr11. Flux analysis of the nitration of Tyr34 by PN-CO2 showed that the nitration rate coincided with the kinetics of the reaction of PN with CO2. These kinetics and the 20-fold increase in the efficiency of tyrosine nitration in the presence of CO2 suggest a specific role for the carbonate radical anion (•CO3-) in MnSOD nitration by PN. We also observed that the nitration of Tyr34 caused inactivation of the enzyme, while nitration of Tyr9 and Tyr11 did not interfere with the superoxide dismutase activity. The loss of MnSOD activity upon Tyr34 nitration implies that the responsible reagent in vivo is peroxynitrite, acting either directly or through the action of •CO3-.

Reactive Oxygen and Nitrogen Species in Steatotic Hepatocytes: A Molecular Perspective on the Pathophysiology of Ischemia-Reperfusion Injury in the Fatty Liver

“…The consequences hereof likely contribute to the susceptibility of steatotic hepatocytes to IR injury, especially when these modifications affect antioxidant enzymes. Tyrosine nitration and histidine oxidation, for instance, are key events in the peroxynitrite-induced inactivation of MnSOD (193) and copper-zinc-SOD (5), respectively. The functional loss of these enzymes was found to contribute to the reduced antioxidative capacity in NAFLD livers (209), which is likely to affect cell viability during IR (section ''Pathophysiological implications of ROS/RNS in NAFLD and IR'').…”

Copper-Catalyzed Tyrosine Nitration

“…• NO 2 can be generated via several mechanisms, including the oxidation of • NO by oxygen, 12 the decomposition of ONOO À , 7,11,13 and the oxidation of nitrite (NO 2 À ) by hydrogen peroxide (H 2 O 2 ) catalyzed with peroxidases.…”

Proteomics quantification of protein nitration

“…Similarly, elevated levels of nitration are observed in cardiomyopathy, cardiovascular disease, atherosclerosis (Leeuwenburgh et al 1997, Turko and Murad 2002, Ungvari et al 2005, Peluffo and Radi 2007, Upmacis 2008, Lee et al 2009a, Surmeli et al 2010, arthritis (Sandhu et al 2003 ), diabetes (Turko et al 2003 ), cancer (Azad et al 2008 ), and aging (Pacher et al 2007, Marshall et al 2013. It can be inferred from the range of disorders affected by this PTM that the immune system is a major target of RNS.…”