Evidence for the Participation of Cys₅₅₈ and Cys₅₅₉ at the Active Site of Mercuric Reductase

Mercuric reductase, with FAD and a reducible disulfide at the active site, catalyzes the two-electron reduction of Hg(II) by NADPH. Addition of reducing equivalents rapidly produces a spectrally distinct EH₂ form of the enzyme containing oxidized FAD and reduced active site thiols. Formation of EH₂ has previously been reported to require only 2 electrons for reduction of the active site disulfide. We present results of anaerobic titrations of mercuric reductase with NADPH and dithionite showing that the equilibrium conversion of oxidized enzyme to EH₂ actually requires 2 equiv of reducing agent or 4 electrons. Kinetic studies conducted both at 4 °C and at 25 °C indicate that reduction of the active site occurs rapidly, as previously reported [Sahlman, L., & Lindskog., S. (1983) Biochem. Biophys. Res. Commun. 117, 231–237]; this is followed by a slower reduction of another redox group via reaction with the active site. Thiol titrations of denatured E_ox and EH₂ enzyme forms show that an additional disulfide is the group in communication with the active site.[¹⁴C] Iodoacetamide labeling experiments demonstrate that the C-terminal residues, Cys₅₅₈ and Cys₅₅₉,are involved in this disulfide. The fluorescence, but not the absorbance, of the enzyme-bound FAD was found to be highly dependent on the redox state of the C-terminal thiols. Thus, Eox with Cys₅₅₈ and Cys₅₅₉ as thiols exhibits less than 50% of the fluorescence of E_ox where these residues are present as a disulfide, indicating that the thiols remain intimately associated with the active site. Initial velocity measurements show that the auxiliary disulfide must be reduced before catalytic Hg(II) reduction can occur, consistent with the report of a preactivation phenomenon with NADPH or cysteine [Sandstrom, A., & Lindskog., S.(1987) Eur, J. Biochem. 164, 243–249]. A modified minimal catalytic mechanism is proposed as well as several chemical mechanisms for the Hg(II) reduction step.