Rapid‐scan stopped‐flow studies of the pH dependence of the reaction between mercuric reductase and NADPH

The reaction of NADPH with the flavoenzyme mercuric reductase has been studied by rapid‐scan stopped‐flow spectrophotometry at 5°C in the pH range 5.1–9.5. An intermediate formed within the dead time of the apparatus, and proposed to be an NADPH complex of oxidized enzyme, has an almost pH‐independent spectrum. At pH 5.1 the formation of this species is followed by a rapid bleaching ( k = 145 s −1 ) of the main flavin absorption band at 455 nm concomitantly with an absorbance increase around 395 nm. This process, which has a kinetic hydrogen isotope effect of 2.4 becomes less prominent at higher pH values and is not detectable above pH 7. It is suggested that this process includes the formation of a covalent thiol‐flavin C‐4a derivative stabilized by protonation of the active site. In the presence of an excess of NADPH, the final product of the reaction is probably an NADPH complex of two‐electron‐reduced enzyme, but below pH 6 the final spectrum becomes less intense suggesting a partial formation of four‐electron‐reduced enzyme. The spectral changes observed above pH 7 are nearly independent of pH. The first measurable step ( k = 48 s −1 at pH 9.5) is thought to include the formation of an NADP + complex of two‐electron‐reduced enzyme, while the final step ( k = 6.3 s −1 at pH 9.5) results in the above‐mentioned NADPH complex with two‐electron‐reduced enzyme. A minimal kinetic scheme rationalizing the observed pH dependence of the reaction and the observed isotope effects is presented.