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 degrees 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.
D000693:Anaerobiosis; D002850:Chromatography, Gel; D003201:Computers; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000032:analysis
D013438:Sulfhydryl Compounds; D009249:NADP; D003903:Deuterium; D010088:Oxidoreductases; C021551:mercuric reductase