REDUCTIVE AND OXIDATIVE HALF-REACTIONS OF GLUTATHIONE-REDUCTASE FROM ESCHERICHIA-COLI

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH and has a redox active disulfide and an FAD cofactor in each monomer. In the reductive half-reaction, FAD is reduced by NADPH and electrons pass from the reduced flavin to the redox active disulfide. The oxidative half-reaction is dithiol-disulfide interchange between t he enzyme dithiol and glutathione disulfide. We have investigated the reductive and oxidative half-reactions using wild-type glutathione red uctase from Escherichia coli and in an altered form of the enzyme in w hich the active site acid-base catalyst, His(439), has been changed to an alanine residue (H439A). H439A has 0.3% activity in the NADPH/GSSG assay. The replacement affects both the oxidative half-reaction, as e xpected, and the reductive half-reaction-specifically, the passage of electrons from reduced flavin to the disulfide. Reduction of H439A by NADPH allows direct observation of flavin reduction. The NADPH-FAD cha rge transfer complex is formed in the dead time. Reduction of FAD, at a limiting rate of 250 s(-1), is observed as a decrease at 460 nm and an increase at 670 nm (FADH(-)-NADP(+) charge transfer). Subsequent pa ssage of electrons from FADH(-) to the disulfide (increase at 460 nm a nd a decrease at 670 nm) is very slow (6-7 s(-1)) and concentration in dependent in H439A. The monophasic oxidative half-reaction is very slo w, as expected for reduced H439A. The limiting rate of the reductive h alf-reaction in wild-type enzyme is independent of the NADPH concentra tion and determined to be 110 s(-1), while the limiting rate of the ox idative half-reaction has been estimated as 490 s(-1), and is dependen t on the glutathione disulfide concentration. Thus, the acid-base cata lyst participates in the disulfide reduction step by stabilizing the n ascent thiolate and in the oxidative half-reaction by protonating the dissociating glutathione thiolate anion. Both roles are consistent wit h proposed mechanisms.