S-Nitrosoglutathione (GSNO) represents a major transport form in nitric 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). Catalyzing the reaction NADPH+GSSG+H(+)-->NADP(+) + 2 GSH, the dimeric flavoprotein GR is the central enzyme of the glutathione redox metabolism. GSNO was found to inhibit crystalline erythrocyte GR in two ways: (a) as a reversible inhibitor GSNO is competitive with glutathione disulfide (GSSG), the Ki being appr. 0.5 mM; (b) as an irreversible inhibitor; after 1 h (3 h) incubation with 1 mM GSNO, GR (2.5 U/ml, representing intraerythrocytic concentrations) was inhibited by 70% (90%). This inhibition depended on the presence of NADPH and could not be reversed by dilution nor by reducing agents. Absorption spectra indicate that the charge-transfer interaction between Cys63 and the flavin is abolished by this modification. In a GR sample inhibited by 90% with GSNO, the Km values for the substrates GSSG and NADPH were not significantly changed nor did the modification induce oxidase activity of the enzyme. GSNO was found not to be a substrate in the forward reaction of GR. This implies that GSNO is not accounted for by methods which employ GR for determining total glutathione. Incubating isolated GR for 60 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 GSNO to the enzyme. GSNO inhibition patterns comparable to purified authentic GR were obtained for purified recombinant GR, a GR mutant lacking the 15 N-terminal amino acids including Cys2, and for the enzyme present in diluted fresh haemolysates (0.02 U/ml); in concentrated haemolysates the inhibition was less pronounced. GR of intact erythrocytes was 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-modified GR for X-ray diffraction analysis.