Peroxynitrite Modification of Protein Thiols:  Oxidation, Nitrosylation, and S-Glutathiolation of Functionally Important Cysteine Residue(s) in the Sarcoplasmic Reticulum Ca-ATPase

Abstract

Skeletal muscle contraction and relaxation is efficiently modulated through the reaction of reactive oxygen-nitrogen species with sarcoplasmic reticulum protein thiols in vivo. However, the exact locations of functionally important modifications are at present unknown. Here, we determine by HPLC-MS that the modification of one (out of 24) Cys residue of the sarcoplasmic reticulum (SR) Ca-ATPase isoform SERCA1, Cys(349), by peroxynitrite is sufficient for the modulation of enzyme activity. Despite the size and nature of the SR Ca-ATPase, a 110 kDa membrane protein, identification and quantitation of Cys modification was achieved through labeling with 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABMI) and/or N-(2-iodoethyl)trifluoroacetamide (IE-TFA) followed by an exhaustive tryptic digestion and on-line HPLC-UV-electrospray MS analysis. The reaction with IE-TFA generates aminoethylcysteine, a new trypsin cleavage site, which allows the production of specific peptide fragments that are diagnostic for IE-TFA labeling, conveniently identified by mass spectrometry. Exposure of the SR Ca-ATPase to low concentrations (0.1 mM) of peroxynitrite resulted in the fully reversible chemical modification of Cys at positions 344, 349, 471, 498, 525, and 614 (nitrosylation of Cys(344) and Cys(349) was seen), whereas higher concentrations of peroxynitrite (0.45 mM) additionally affected Cys residues at positions 636, 670, and 674. When the SR Ca-ATPase was exposed to 0.45 mM peroxynitrite in the presence of 5.0 mM glutathione (GSH), thiol modification became partially reversible and S-glutathiolation was detected for Cys residues at positions 344, 349, 364, 498, 525, and 614. The extent of enzyme inactivation (determined previously) quantitatively correlated with the loss of labeling efficiency (i) of a single Cys residue and (ii) of the tryptic fragment containing both Cys(344) and Cys(349). Earlier results had shown that the independent selective modification of Cys(344) is functionally insignificant [Kawakita, M., and Yamashita, T. (1987) J. Biochem. (Tokyo) 102, 103-109]. Thus, we conclude that modification of only Cys(349) is responsible for the modulation of the SR Ca-ATPase activity by peroxynitrite.

Inhibition of Skeletal Muscle S1-Myosin ATPase by Peroxynitrite

“…Briefly, oxidation of Cys 707 produces an increase of nonphysiological Ca 2+ -ATPase myosin activity, whereas oxidation of both reactive cysteines inhibits all of the myosinATPase activities (45). However, peroxynitrite-induced oxidation of highly reactive cysteines is one of the most established protein-chemical modifications induced by this reagent (12,16,50). Figure 3A shows that exposure to SIN-1 results in inhibition of K + /EDTA-ATPase and Ca 2+ -ATPase activities of S1 with IC 50 values close to that found for the inhibition of F-actin-stimulated Mg 2+ -ATPase activity and (9), or 25 mM bicarbonate (4), and after 2 h, the aliquots were pooled for assaying the activity as indicated in the legend of Figure 1A.…”

“…Protein-cysteine oxidation by peroxynitrite has been shown to cause the inhibition of sarcoplasmic-reticulum Ca 2+ ATPase (15,16). In addition, peroxynitrite can modify proteins by acting through radicals generated during its spontaneous decomposition in biochemical buffers and biological fluids, particularly, HO‚ and NO 2 ‚, a reaction pathway that is potentiated by the presence of bicarbonate, which upon reaction with peroxynitrite leads to the production of carboxylate radical CO 3 ‚ - (17,18).…”

Superoxide as a Messenger of Endothelial Function

“…PN generated from NO and • O 2 -can react with Tyr or Tyr-containing proteins to form 3-nitrotyrosine (3-NT) (Crow and Beckman, 1995;Beckman and Kopppenol, 1996;Viner et al, 1999) but in general the required concentrations are higher than expected to occur in vivo. Pfeiffer and Mayer (Pfeiffer et al, 1998;2001a;2001b) have even questioned the significance of PN as a cellular nitrating agent and have proposed nitrite / hydrogen peroxide as an alternative pathway with myeloperoxidase as a catalyst (Brennan et al, 4-2 2002;van Dalen et al, 2000).…”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…The amount and complexity of nitrosating species produced in vivo is further complicated by the ability of NO to rapidly react with superoxide to form peroxynitrite (7 x 10 9 M -1 .s -1 ) [39]. Peroxynitrite can protonate and then liberate • NO 2 which can combine with NO to generate nitrosating N 2 O 3 [40]. However, peroxynitrite can also induce disulfide formation in proteins independently of S-nitrosothiol intermediates [41].…”

“…There are 7 proposed sites of S-nitrosation within SERCA [40]. Selective inhibition of neuronal NOS in rat hearts resulted in a significant decrease in the contractile response to isoproterenol, with a corresponding 50% decrease in S-nitrosation of SERCA [124].…”