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, Cys₃₄₉, 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 Cys₃₄₄ and Cys₃₄₉ 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 Cys₃₄₄ and Cys₃₄₉. Earlier results had shown that the independent selective modification of Cys₃₄₄ is functionally insignificant [Kawakita, M., and Yamashita, T. (1987) J. Biochem. (Tokyo)102, 103−109]. Thus, we conclude that modification of only Cys₃₄₉ is responsible for the modulation of the SR Ca-ATPase activity by peroxynitrite.