Rapid‐scan stopped‐flow studies of the flavoenzyme mercuric reductase during catalytic turnover

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Abstract
Time‐resolved absorption spectra of the FAD‐containing enzyme mercuric reductase were recorded during the catalytic reaction at 25°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‐NADP+ 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.