Soulère L, Claparols C, Périé J, Hoffmann P
Groupe de Chimie Organique Biologique, Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique, UMR 5068, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 4, France.
Biochem J. 2001 Dec 15;360(Pt 3):563-7. doi: 10.1042/0264-6021:3600563.
The peroxynitrite anion is a potent oxidizing agent, formed by the diffusion-limited combination of nitric oxide and superoxide, and its production under physiological conditions is associated with the pathologies of a number of inflammatory and neurodegenerative diseases. Nitration of Escherichia coli iron superoxide dismutase (Fe-SOD) by peroxynitrite was investigated, and demonstrated by spectral changes and electrospray mass spectroscopic analysis. HPLC and mass studies of the tryptic digests of the mono-nitrated Fe-SOD indicated that tyrosine-34 was the residue most susceptible to nitration by peroxynitrite. Exclusive nitration of this residue occurred when Fe-SOD was exposed to a cumulative dose of 0.4 mM peroxynitrite. Unlike with human Mn-SOD, this single modification did not inactivate E. coli Fe-SOD at pH 7.4. When Fe-SOD was exposed to higher concentrations of peroxynitrite (7 mM), eight tyrosine residues per subunit of the protein, of the nine available, were nitrated without loss of catalytic activity of the enzyme. The pK(a) of nitrated tyrosine-34 was determined to be 7.95+/-0.15, indicating that the peroxynitrite-modified enzyme appreciably maintains its protonation state under physiological conditions.
过氧亚硝酸根阴离子是一种强氧化剂，由一氧化氮和超氧化物通过扩散限制结合形成，其在生理条件下的产生与多种炎症和神经退行性疾病的病理过程相关。研究了过氧亚硝酸根对大肠杆菌铁超氧化物歧化酶（Fe-SOD）的硝化作用，并通过光谱变化和电喷雾质谱分析进行了证明。对单硝化Fe-SOD的胰蛋白酶消化产物进行的HPLC和质谱研究表明，酪氨酸-34是最易受过氧亚硝酸根硝化的残基。当Fe-SOD暴露于累积剂量为0.4 mM的过氧亚硝酸根时，该残基发生了专一性硝化。与人类锰超氧化物歧化酶不同，这种单一修饰在pH 7.4时并未使大肠杆菌Fe-SOD失活。当Fe-SOD暴露于更高浓度的过氧亚硝酸根（7 mM）时，该蛋白质每个亚基的九个可用酪氨酸残基中有八个被硝化，但酶的催化活性并未丧失。经测定，硝化酪氨酸-34的pK(a)为7.95±0.15，这表明过氧亚硝酸根修饰的酶在生理条件下能明显保持其质子化状态。
