Original ContributionInactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide
Introduction
Superoxide radical (O2−) is formed in vivo through the one-electron reduction of molecular oxygen by flavoproteins like xanthine oxidase [1], [2], the mitochondrial respiratory chain [3], activation of NADPH oxidase [4], [5], and oxidation of quinols [6]. Under normal conditions, superoxide formation rates are assumed to be as high as 34 μM/min in mitochondria [3], [7]. However, intracellular superoxide steady-state concentrations are kept very low (∼ 10−10 M) [3], [7], [8] due to the action of superoxide dismutases (SOD), CuZnSOD and MnSOD, which catalyze the dismutation of superoxide to hydrogen peroxide and molecular oxygen with rate constants of 1–2 × 109 M−1 s−1 [9], [10], [11]. Despite its free radical nature, superoxide is in general not too reactive; its principal targets are [4Fe–4S]-containing dehydratases, such as aconitase [8], [12]. However, superoxide can react with nitric oxide (NO), an inorganic radical that is also formed in vivo by the action of nitric oxide synthase (NOS), at nearly diffusion-limited rates (0.5–1.9 × 1010 M−1 s−1) to form peroxynitrite [13], [14], [15].
Peroxynitrite1 is a potent one- and two-electron oxidant (E°′(ONOOH, H+/NO2, H2O) = 1.6–1.7 V and E°′(ONOOH, H+/NO2−, H2O) = 1.3–1.37 V) [16], [17] and a nitrating species. This molecule can react directly with several targets such as thiols [18], protein metal centers [19], carbon dioxide [20], [21], and selenium compounds [22]. At pH 7.4, peroxynitrite is mostly in its anionic form but in equilibrium with its conjugated acid, peroxynitrous acid (pKA6.8) which can then homolyze at a rate of 0.9 s−1 at pH 7.4, 37°C [18], [23], forming nitrogen dioxide (NO2) and hydroxyl radical (OH) in a ∼ 30% yield [24], [25]. These radicals participate in peroxynitrite-dependent toxicity, being involved in tyrosine nitration [26], [27] and lipid peroxidation [28], [29] processes.
Nitration and oxidation of free tyrosine by simultaneous fluxes of nitric oxide and superoxide have been related to peroxynitrite formation by several authors [30], [31], [32], [33], [34]. In one of those studies [30], the maximum yield of nitration was achieved at equal rates of superoxide and nitric oxide formation, generating a “bell-shaped” curve in which at high nitric oxide/superoxide or superoxide/nitric oxide ratios, tyrosine nitration was inhibited. In recent theoretical reports, including one of our group [35], [36], this bell-shaped profile was completely lost, due to the incorporation of biologically relevant events such as nitric oxide diffusion and superoxide-catalyzed dismutation. Moreover, nitration became responsive to increases in nitric oxide and superoxide rates, irrespective of the relative superoxide/nitric oxide flux ratio and in agreement with in vivo observations (for a critical analysis see [27]).
Under physiological conditions most of the superoxide produced in the cell dismutates in the reaction catalyzed by SOD [37] and only marginal basal levels of tyrosine nitration are observed [38]. However, under pathological conditions when nitric oxide levels increase due to the expression of inducible NOS (iNOS) or overactivation of endothelial NOS (eNOS) and neuronal NOS (nNOS) [39], biological markers of ONOO− formation, such as 3-nitrotyrosine are substantially augmented [38], [40], [41], [42]. Thus, nitric oxide reaction with superoxide occurs despite the SOD-catalyzed dismutation of superoxide posing a “kinetic dilemma” whose resolution can be aided by specific in vitro and in silico approaches. Moreover, experimental evidence in vivo indicates that MnSOD is nitrated and inactivated under pathological conditions [38], [41], [43], [44], [45], [46], with nitration at the active site tyrosine-34 being mainly responsible for the loss of activity [38], [47], [48]. Both MnSOD and CuZnSOD react directly with peroxynitrite with second-order rate constants of 2.5 × 104 and 9.4 × 103 M−1 s−1 per monomer, respectively [49], [50]. Both enzymes are, also, inactivated by peroxynitrite [47], [50], [51] with tyrosine nitration the main modification observed in MnSOD, which has three tyrosine residues susceptible to nitration (Y-45, Y-193, and the critical for activity Y-34) [48]. In CuZnSOD histidinyl radical formation was observed after exposure to peroxynitrite [50] while tyrosine nitration cannot occur due to the lack of this residue in the primary structure of this enzyme. In both cases, a direct reaction between peroxynitrite and the metal centers of the enzymes is proposed as mainly responsible for their site-specific inactivation and, as for other Lewis acids, it has been proposed that peroxynitrite reacts with the metal center forming an adduct that homolyzes to yield nitrogen dioxide and the corresponding oxyradical [49], [50]. However, it is still not clear, in the case of MnSOD, how nitrogen dioxide arising from the reaction of peroxynitrite with manganese ions can lead to tyrosine nitration in the presence of biologically relevant levels of glutathione (5–10 mM) [36] which readily (k = 2 × 107 M−1 s−1) scavenges this oxidant [52].
Herein, we exposed human superoxide dismutases (CuZnSOD and MnSOD) to simultaneous fluxes of nitric oxide and superoxide, and studied their inactivation along with the formation of protein radicals and 3-nitrotyrosine. This approach provides a way to prove that biological fluxes of nitric oxide can outcompete superoxide from the enzymatic dismutation to form peroxynitrite, in a system that more closely resembles a biological condition. Moreover, the occurrence of nitration in the presence of relevant levels of glutathione was investigated.
Section snippets
Chemical reagents
Spermine NONOate was obtained from Alexis, xanthine oxidase from Calbiochem, and catalase from Fluka. Double-distilled 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was generously provided by Dr. R. Mason (NIEHS, NC). Hydrogen peroxide was from J. T. Baker and its concentration was determined from the absorbance at 240 nm (ε = 43.6 M−1 cm−1) [53]. All other reagents were from Sigma. Peroxynitrite solutions were prepared from acidified hydrogen peroxide and sodium nitrite as described [54]. Hydrogen
CuZnSOD and MnSOD are inactivated by fluxes of nitric oxide and superoxide
Exposure of CuZnSOD (5 μM, dimer) and MnSOD (5 μM, tetramer) to simultaneous equimolar fluxes of nitric oxide (JNO) and superoxide (JO2−) (10 μM/min) led to a time-dependent inactivation of both enzymes (Fig. 1A). Total inactivation was not achieved during the times of exposure that were probed for both enzymes, with a maximum inactivation of ∼ 60% being achieved at 30 min of incubation. The exposure to only one of the fluxes (either superoxide or nitric oxide) did not affect the activity of the
Discussion
SOD inactivation by peroxynitrite in vitro is an extensively reported event [41], [47], [48], [49], [50], [51], and firm evidence exists for the occurrence of the reaction in vivo for MnSOD [38], [43], [44], [45], [46]. Yet, the actual formation of peroxynitrite and its reaction with SOD are rather counterintuitive concepts, because superoxide reacts with the enzyme at close to diffusion-limited rates and SOD concentration is relatively high (4–40 μM CuZnSOD and 1–30 μM MnSOD [49], [73]).
Note added in proof
A report has just indicated that aerobic incubation of E. coli MnSOD with relatively high concentrations of nitric oxide can yield nitroxyl anion and secondarily peroxynitrite that can cause nitration and inactivation of the enzyme (Filipovic M.R., Stanic D., Raicevic S., Spasic M. and Niketic V. Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide. Free Radical Research. 42: 62-72 (2007)).
Acknowledgments
We thank Dr. Mónica Marín (Universidad de la República) for kindly providing an anti-human CuZn SOD antibody. V.D. is partially supported by a Ph.D. fellowship from PEDECIBA (Programa de Desarrollo de Ciencias Básicas, Uruguay). This work was supported by grants from the Howard Hughes Medical Institute to R.R., Comisión Sectorial de Investigación Científica, Universidad de la República to B.A., and Fondo Clemente Estable to C.Q. R.R. is a Howard Hughes International Research Scholar.
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