One-Electron Reduction of Chromate by NADPH-dependent Glutathione Reductase

"Electron spin resonance (ESR) measurements provide evidence for the formation of Cr(V) intermediates in the enzymatic reduction of Cr(VI) by glutathione reductase (GSSG-R) in the presence of NADPH, indicating an initial single-electron transfer step in the reduction mechanism. Depending on the pH, at least two different Cr(V) species are generated which are relatively long-lived. In addition, we have detected the hydroxyl (·OH) radical formation during the GSSG-R catalyzed reduction of Cr(VI) by spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and a-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the ·OH radical and Cr(V) formation, indicating that the ~ - is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the ·OH radical formation, indicating the involvement of hydrogen peroxide (H20 2) in the reaction mechanism. Addition of H20i suppresses Cr(V) and enhances the ·OH radical fonnation. Measurements involving N-ethylmaleimide show that the Cr(V) species, produced enzymatically by the reduction of Cr(VI) by GSSG-R, react with H2O2 to generate ·OH radicals, which might participate in the initiation of Cr(VI) carcinogenicity.INTRODUCTIONC(VI) compounds have been found to exert serious toxic and carcinogenic effects on humans and animals [1-4) and to cause mutations in bacteria and transformation of mammalian cells [5-8). In contrast, most Cr(lll) compounds are relatively nontoxic, noncarcinogenic, and nonmutagenic [9, 10]. Cr(VI) and Cr(lll) oxidation states are different in their metabolic pathways: Cr(VI) ions are rapidly transported across cellular membranes [2, 11-13), while Cr(lll) moieties do not easily penetrate cells and are not oxidized by cellular constituents [3]. Since it is known that the reduction of Cr(Vl) is required for its reaction with DNA [2], the molecular mechanisms for the intracellular Cr(VI) reduction have been the focus of studies during the past decade but the details are still not understood."