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 ({dot operator}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 α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the {dot operator}OH radical and Cr(V) formation, indicating that the O₂^- is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the {dot operator}OH radical formation, indicating the involvement of hydrogen peroxide (H₂O₂) in the reaction mechanism. Addition of H₂O₂ suppresses Cr(V) and enhances the {dot operator}OH radical formation. 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 {dot operator}OH radicals, which might participate in the initiation of Cr(VI) carcinogenicity.