Characterization of the covalent mercury (II)-NADPH complex

Mercury (II) forms a covalent, 1:1 complex with NADPH which is indicated by the disappearance of the chromophore at 340 nm. With the use of NMNH as an analogue, the spectral changes were attributed to the reaction of Hg(II) with the nicotinamide ring. This reaction is reversed by the addition of sulfhydryl groups or other metal-chelating agents. The structure of the Hg(II) . NADPH complex was determined with 1H and 13C NMR spectroscopy and homonuclear decoupling and was shown to have Hg covalently attached to C-5 of the nicotinamide ring. From these results, we propose that Hg(II) reacts with the double bond between C-5 and C-6 to form a mercuronium ion which is opened by the nucleophilic attack of water on C-6. Two conformations of the Hg(II) . NADPH complex were detected, corresponding to the formation of the mercuronium ion on either the a or b side of the nicotinamide ring. Fluorescence quenching studies with the Hg(II) . NADPH complex and the flavoenzyme mercuric reductase showed that the complex could effectively bind (Kd = 2.3 microM), but could not reduce the enzyme.