The reaction between NADPH and mercuric reductase from Pseudomonas aeruginosa.
The reaction between the FAD-containing enzyme, mercuric reductase, and its reducing substrate, NADPH, has been studied at 5 degrees C, pH 7.3, by rapid-scan and fixed-wavelength stopped-flow techniques with the aim to characterize reaction intermediates. With an excess of NADPH the spectral changes observed in rapid-scan experiments can be described by a simple kinetic model, A----B----C----D, where A, B, C, and D represent distinct spectral species. The first step is virtually complete within the dead time of the apparatus. It is associated with a 16% bleaching of flavin absorbance and the appearance of a very broad charge-transfer band (epsilon max = 1.9 mM-1 cm-1) centered near 600 nm. The second step (k = 43 s-1) involves a further small bleaching of flavin absorbance and intensification of the charge-transfer band (epsilon max = 3.3 mM-1 cm-1) which becomes centered near 580 nm. The third step (k = 8 s-1) involves an intensification and a blue shift of the main FAD absorption band. Concurrently, the charge-transfer band increases in intensity and becomes centered near 530 nm (epsilon 530 = 5.0 mM-1 cm-1). While the first two steps involve 1 mol of NADPH per mol of FAD, the third step requires a second equivalent of NADPH. A 2-electron-reduced enzyme (EH2) can be obtained by treatment of the oxidized enzyme (E) with dithioerythritol. Addition of 1 eq of NADP+ to dithioerythritol-generated EH2 gives rise to a spectral species similar to the kinetic intermediate C, while the addition of 1 eq of NADPH gives rise to a spectral species similar to D. It is proposed that B largely corresponds to an E-NADPH complex, C to an EH2-NADP+ complex, and D to an EH2-NADPH complex.
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013053:Spectrophotometry; D013816:Thermodynamics
D009249:NADP; D010088:Oxidoreductases; C021551:mercuric reductase