A stopped-flow study of the reaction between mercuric reductase and NADPH

The reaction of the FAD-containing enzyme, mercuric reductase, with NADPH has been studied by stopped-flow kinetic methods at 25 degrees C, pH 7.3. The results suggest that the reaction involves at least three steps. The first step is very rapid and is essentially complete within the dead time of the stopped-flow apparatus. This step is associated with decreasing absorbances at 340 nm (NADPH) and 455 nm (FAD), whereas there is little formation of the absorbance at 530 nm characterizing 2-electron-reduced enzyme subunits (EH2). The second step involves an increase of the absorbance at 530 nm. The third step results in an increase of the intensity of the long-wavelength band and a change of its shape. A second equivalent of NADPH per FAD is required for this step. It is proposed that the product is an EH2-NADPH complex. In addition to these rapid steps, slow absorbance changes are also observed.