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 the enzyme dithiol and glutathione disulfide. We have investigated the reductive and oxidative half-reactions using wild-type glutathione reductase from Escherichia coli and in an altered form of the enzyme in which the active site acid-base catalyst, His439, 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 expected, 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 charge 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 passage of electrons from FADH- to the disulfide (increase at 460 nm and a decrease at 670 nm) is very slow (6-7 s-1) and concentration independent in H439A. The monophasic oxidative half-reaction is very slow, as expected for reduced H439A.(ABSTRACT TRUNCATED AT 250 WORDS)