The biological signal molecule nitric oxide (NO) exists in a free and carrier-bound form. Since the structure of the carrier is likely to influence the interaction of NO with macromolecular targets, we assessed the interaction of a dinitrosyl-iron-dithiolate complex carrying different thiol ligands with glutathione reductase. The enzyme was irreversibly inhibited by dinitrosyl-iron-di-L-cysteine and dinitrosyl-iron-di-glutathione in a concentration- and time-dependent manner (IC50 30 and 3 microM, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a Ki of 14 microM, and a k3 of 1.3 x 10(-3) s-1. A participation of catalytic site thiols in the inhibitory mechanism was indicated by the findings that only the NADPH-reduced enzyme was inhibited by dinitrosyl-iron complex and that blockade of these thiols by Hg2+ afforded protection against irreversible inhibition. This inhibition was not accompanied by formation of a protein-bound dinitrosyl-iron complex and/or S-nitrosation of active site thiols (Cys-58 and Cys-63). However, one NO moiety exhibiting an acid lability similar to a secondary N-nitrosamine was present per mol of inhibited monomeric enzyme. These findings suggest specifically N-nitrosation of glutathione reductase as a likely mechanism of inhibition elicited by dinitrosyl-iron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO+ transfer to the ligand-binding protein.