INHIBITION OF HUMAN GLUTATHIONE-REDUCTASE BY S-NITROSOGLUTATHIONE

S-Nitrosoglutathione (GSNO) represents a major transport form of nitri c oxide (NO) in biological systems. Since NO and GSNO have been shown to modulate the function of various proteins, we studied the influence of GSNO and other NO donors on human glutathione reductase (GR). Cata lyzing the reaction NADPH + GSSG + H+ --> NADP(+) + 2 GSH, the dimeric flavoprotein GR is the central enzyme of the glutathione redox metabo lism. GSNO was found to inhibit crystalline erythrocyte GR in two ways : (a) as a reversible inhibitor GSNO is competitive with glutathione d isulfide (GSSG), the K, being appr, 0.5 mM; (b) as an irreversible inh ibitor; after 1 h (3 h) incubation with 1 mM GSNO, GR (2.5 U/ml, repre senting intraerythrocytic concentrations) was inhibited by 70% (90%). This inhibition depended on the presence of NADPH and could not be rev ersed by dilution nor by reducing agents. Absorption spectra indicate that the charge-transfer interaction between Cys63 and the flavin is a bolished by this modification. In a GR sample inhibited by 90% with GS NO, the K-m values for the substrates GSSG and NADPH were not signific antly changed nor did the modification induce oxidase activity of the enzyme. GSNO was found not to be a substrate in the forward reaction o f GR. This implies that GSNO is not accounted for by methods which emp loy GR for determining total glutathione. Incubating isolated GR for 6 0 min with other NO donors, namely 1 mM sodium nitroprusside or 1 mM S -nitroso-N-acetyl-DL-penicillamine (SNAP), resulted in only 25% and 10 % inhibition, respectively. This attests to a specific affinity of GSN O to the enzyme. GSNO inhibition patterns comparable to purified authe ntic GR were obtained for purified recombinant GR, a GR mutant lacking the 15 N-terminal amino acids including Cys2, and for the enzyme pres ent in diluted fresh haemolysates (0.02 U/ml); in concentrated haemoly sates the inhibition was less pronounced, GR of intact erythrocytes wa s not affected when exposed to GSNO in the medium. Our results suggest that the irreversible inhibition of GR by GSNO involves nitrosylation of Cys63 and/or Cys58 at the catalytic site of the enzyme. To further investigate the mechanism of inactivation we have crystallized GSNO-m odified GR for X-ray diffraction analysis.