Dinitrosyl-Dithiol-Iron Complexes, Nitric Oxide (NO) Carriers In Vivo, as Potent Inhibitors of Human Glutathione Reductase and Glutathione- S-Transferase

Abstract

Human glutathione reductase (GR) and rat liver glutathione- S-transferases (GSTs) had been shown to be inhibited by the nitric oxide (NO) carrier S-nitroso-glutathione (GSNO). We have now extended these studies by measuring the effects of dinitrosyl-iron complexed thiols (DNIC-[RSH] 2) on human GR, GST and glutathione peroxidase. DNIC-[RSH] 2 represent important transport forms of NO but also of iron ions and glutathione in vivo. Human GR was found to be inhibited by dinitrosyl-iron-di-glutathione (DNIC-[GSH] 2) and dinitrosyl-iron-di- l-cysteine (DNIC-Cys 2) in two ways: both compounds were competitive with glutathione disulfide (GSSG), the inhibition constant ( K i) for reversible competition of DNIC-[GSH] 2 with GSSG being approximately 5 μM; preincubating GR for 10 min with 4 μM DNIC-[GSH] 2 and 40 μM DNIC-Cys 2, respectively, led to 50% irreversible enzyme inactivation. More than 95% GR inactivation was achieved by incubation with 36 μM DNIC-[GSH] 2 for 30 min. This inhibition depended on the presence of NADPH. Absorption spectra of inhibited GR showed that the charge-transfer interaction between the isoalloxazine moiety of the prosthetic group flavin adenine dinucleotide (FAD) and the active site thiol Cys63 is disturbed by the modification. Cys 2 and FAD could be ruled out as sites of the modification. Isolated human placenta glutathione-S-transferase and GST activity measured in hemolysates were also inhibited by DNIC-[GSH] 2. This inhibition, however, was reversible and competitive with reduced glutathione, the K i being 20 nM. The inhibition of GST induced by GSNO was competitive with reduced glutathione (GSH) ( K i = 180 μM) and with the second substrate of the reaction, 1-chloro-2,4,-dinitrobenzene ( K i = 170 μM). An inhibition of human glutathione peroxidase by GSNO or DNIC-[RSH] 2 was not detectable. Inactivation of GR by DNIC-[GSH] 2 is by two orders of magnitude more effective than modification by GSNO; this result and the very efficient inhibition of GST point to a role of DNIC-[RSH] 2 in glutathione metabolism.