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 degrees C and at 25 degrees 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 Eox 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 Eox where these residues are present as a disulfide, indicating that the thiols remain intimately associated with the active site.(ABSTRACT TRUNCATED AT 250 WORDS)
Miller
S M
SM
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Moore
M J
MJ
Massey
V
V
Williams
C H
CH
Distefano
M D
MD
Ballou
D P
DP
Walsh
C T
CT
D001665:Binding Sites; D003545:Cysteine; D004220:Disulfides; D004227:Dithionite / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry
D004220:Disulfides; D004227:Dithionite; D009249:NADP; D010088:Oxidoreductases; C021551:mercuric reductase; D003545:Cysteine; D004229:Dithiothreitol