Rapid-scan stopped-flow studies of the flavoenzyme mercuric reductase during catalytic turnover.
Time-resolved absorption spectra of the FAD-containing enzyme mercuric reductase were recorded during the catalytic reaction at 25 degrees C, pH 7.3. With an excess of NADPH over Hg2+ there was a rapid (k = 43 s-1) initial formation of a spectral species similar to that previously assigned to an NADPH complex of two-electron-reduced enzyme, EH2-NADPH. This spectrum persisted during the quasisteady-state phase of the reaction suggesting that EH2-NADPH is a true catalytic intermediate and that the rate of catalysis is limited by the oxidation of EH2-NADPH by Hg2+. Also with an excess of Hg2+ over NADPH a spectrum similar to that of EH2-NADPH was rapidly formed. As the NADPH was exhausted, the spectrum of oxidized enzyme, E, did not reappear but rather a spectrum similar to that previously assigned to an NADP+ complex of two-electron-reduced enzyme, EH2-NADP+. These results suggest that EH2-HADP+ cannot rapidly reduce the Hg2+ substrate. However, eventually all reducing equivalents from NADPH added to oxidized, activated enzyme are utilized for the reduction of Hg2+. A mechanism model is proposed that does not involve the free, oxidized enzyme in the catalytic cycle.
D002384:Catalysis; D003545:Cysteine / Q000494:pharmacology; D008962:Models, Theoretical; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology
D009249:NADP; D010088:Oxidoreductases; C021551:mercuric reductase; D003545:Cysteine