Inhibition of glutathione reductase by dinitrosyl-iron-dithiolate complex.
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.
Boese
M
M
Zentrum der Physiologie, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany.
Keese
M A
MA
Becker
K
K
Busse
R
R
Mülsch
A
A
D000818:Animals; D002384:Catalysis; D002417:Cattle; D004047:Diethyl Pyrocarbonate / Q000378:metabolism / Q000494:pharmacology; D004229:Dithiothreitol / Q000378:metabolism / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors*; D007413:Intestinal Mucosa / Q000201:enzymology; D007501:Iron / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D009249:NADP / Q000378:metabolism; D009589:Nitrogen Oxides / Q000378:metabolism / Q000494:pharmacology*; D015538:Nitrosation; D013438:Sulfhydryl Compounds / Q000378:metabolism / Q000494:pharmacology*
D046911:Macromolecular Substances; D009589:Nitrogen Oxides; D013438:Sulfhydryl Compounds; D009249:NADP; C073661:dinitrosyl iron complex; D007501:Iron; D005980:Glutathione Reductase; D005978:Glutathione; D004047:Diethyl Pyrocarbonate; D004229:Dithiothreitol; D019803:Glutathione Disulfide