Inhibition of myeloperoxidase-mediated protein nitration by tempol: Kinetics, mechanism, and implications

Abstract

Despite the therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetra-methyl-1-piperidinyloxy) and related nitroxides as antioxidants, their effects on peroxidase-mediated protein tyrosine nitration remain unexplored. This posttranslational protein modification is a biomarker of nitric oxide-derived oxidants, and, relevantly, it parallels tissue injury in animal models of inflammation and is attenuated by tempol treatment. Here, we examine tempol effects on ribonuclease (RNase) nitration mediated by myeloperoxidase (MPO), a mammalian enzyme that plays a central role in various inflammatory processes. Some experiments were also performed with horseradish peroxidase (HRP). We show that tempol efficiently inhibits peroxidase-mediated RNase nitration. For instance, 10 M tempol was able to inhibit by 90% the yield of 290 M 3-nitrotyrosine produced from 370 M RNase. The effect of tempol was not completely catalytic because part of it was consumed by recombination with RNase-tyrosyl radicals. The second-order rate constant of the reaction of tempol with MPO compound I and II were determined by stopped-flow kinetics as 3.3 ؋ 10 6 and 2.6 ؋ 10 4 M ؊1 s ؊1 , respectively (pH 7.4, 25°C); the corresponding HRP constants were orders of magnitude smaller. Time-dependent hydrogen peroxide and nitrite consumption and oxygen production in the incubations were quantified experimentally and modeled by kinetic simulations. The results indicate that tempol inhibits peroxidase-mediated RNase nitration mainly because of its reaction with nitrogen dioxide to produce the oxammonium cation, which, in turn, recycles back to tempol by reacting with hydrogen peroxide and superoxide radical to produce oxygen and regenerate nitrite. The implications for nitroxide antioxidant mechanisms are discussed.antioxidant ͉ tyrosine ͉ tyrosine nitration ͉ nitroxides ͉ nitric oxide-derived oxidants

The carbonylation and covalent dimerization of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity is inhibited by the radical scavenger tempol

“…Tempol and other cyclic nitroxides inhibit the peroxidase activity of haem proteins either by reacting with and deactivating higher oxidation intermediates produced during the turnover of these enzymes with H 2 O 2 , such as compounds I and II [32,41,46], or by deactivating substrate-derived radicals [49]. Previously, the effects of nitroxides on the peroxidase activity of SOD1, a copper enzyme, had not been examined, most likely because this activity is quite low and is secondary to the main function of the enzyme [36].…”

Infiltrating T lymphocytes in the kidney increase oxidative stress and participate in the development of hypertension and renal disease

“…In the present set of experiments, tempol treatment during the high-salt period ameliorated hypertension and albuminuria without affecting the infiltration of T cells into the kidney, which remained elevated. Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl) is a small cell-permeable cyclic nitroxide with SOD activity (46); hence, it is capable of catalyzing the conversion of superoxide to hydrogen peroxide, scavenging in this way the O 2 ·Ϫ being produced intracellularly. Therefore, it seems probable that tempol ameliorates hypertension and albuminuria by scavenging the O 2 ·Ϫ that the infiltrating T lymphocytes produce within the kidney.…”

Proteomics in investigation of protein nitration in kidney disease: Technical challenges and perspectives from the spontaneously hypertensive rat

“…This second observation is based on the fact that the SODmimetic TEMPOL and catalase infusion were both necessary in the kidney medulla to improve perfusion and to reduce hypertension; TEMPOL infusion alone was insufficient (Chen, Cowley, & Zou, 2003;Makino et al, 2003). More recently, it has been shown that TEMPOL can inhibit myeloperoxidase (MPO)-mediated nitration of RNase in vitro through reaction with NO : 2 (Vaz & Augusto, 2008). This might suggest that TEMPOL can be put to similar use in vivo.…”