Steady-state and laser flash induced photoreduction of yeast glutathione reductase by 5-deazariboflavin and by a viologen analogue: stabilization of flavin adenine dinucleotide semiquinone species by complexation.

Steady-state and laser flash photolysis techniques have been used to examine the photoreduction of yeast glutathione reductase by the one-electron reduction products of 5-deazariboflavin and the viologen analogue 1,1'-propylene-2,2'-bipyridyl. Steady-state photoreduction of the enzyme with the viologen generates the two-electron-reduced form, whereas photoreduction with deazaflavin generates the anion semiquinone. Flash photolysis indicates that the product of viologen radical reduction is also a semiquinone, suggesting that this species is rapidly further reduced by viologen in the steady-state experiment to form the EH2 enzyme. This reduction is apparently inhibited when deazaflavin is the photoreductant, perhaps due to complexation of the anion semiquinone with deazaflavin. Steady-state experiments demonstrate that complexation of the anion semiquinone with NADP+ also inhibits further reduction. Both one-electron reduction reactions of oxidized glutathione reductase proceed at close to diffusion-controlled rates (second-order rate constants = 10(8)-10(9) M-1 s-1), despite the relatively buried nature of the FAD cofactor. Addition of NADP+ and oxidized glutathione produced no effects on the kinetics of the initial entry of the electron into the enzyme. No kinetic evidence of intramolecular electron transfer involving the FAD and the protein disulfide was obtained during or subsequent to the initial one-electron reduction process. Thus, if this reaction occurs in the semiquinone, it must be quite rapid (k greater than 8000 s-1).