FIG. 2.

Reduction of a flavoprotein (human GR mutant A58/A63) with NADPH and reoxidation by O₂ or by MB in assay buffer at pH 6.9. (A) The black curve shows the original spectrum of the oxidized flavoprotein (8.2 μM) with an absorption maximum at 455 nm. The protein-bound FAD was then reduced with 200 μM NADPH under aerobic conditions to give FADH⁻. This resulted in a decrease in the absorption at 455 nm by 78% (not shown). Subsequent auto-oxidation led to partially reoxidized protein-bound flavin and indirectly to the production of NADP⁺ (gray curve). Here, the characteristic broad peak at around 680 nm represents the complex between protein-bound FADH⁻ and NADP⁺ (33). The original spectrum of oxidized protein-bound flavin (black curve) was recovered after 20 min by auto-oxidation and was 10 times faster by oxidation with 3 μM MB (Fig. 2B). (B). Traces representing the kinetics of FADH⁻ reoxidation by MB. Mutant flavoprotein (9.2 μM) had been incubated under aerobic conditions in GR assay buffer containing 0 to 3 μM MB and monitored at 455 nm in a total volume of 700 μl. The addition of NADPH at 100 μM led to a decrease in the absorption at 455 nm. Reoxidation took place according to the equation protein-bound FADH⁻ + MB⁺→protein-bound FAD + leucoMB, with leucoMB being auto-oxidized instantaneously. The highest rate of absorbance increase was 0.120 min⁻¹. Using a Δɛ of 9.0 mM⁻¹ cm⁻¹ for the difference between reduced flavin and oxidized flavin and correcting for the auto-oxidation of reduced flavin, the second-order rate constant for the reaction of equation 9 was estimated to be 7,500 ± 1,200 M⁻¹ s⁻¹ (15, 33). Solid curve, 3 μM MB; dashed-dotted curve, 0.6 μM MB; dotted curve, 0 μM MB.