Directed mutagenesis of the redox-active disulfide in the flavoenzyme mercuric ion reductase.
Mercuric ion reductase, a flavoenzyme with an active site redox-active cystine, Cys135-Cys140, is an unusual enzyme that reduces Hg(II) to Hg(0) with stoichiometric NADPH oxidation. To probe the catalytic mechanism, we have constructed two active site Cys to Ser mutations by oligonucleotide-directed mutagenesis. The native and the Cys135, Ser140 and Ser135, Cys140 mutant enzymes are expressed on an overproducing plasmid and purified to homogeneity by a one-step procedure in high yield. The optical spectra of the mutant proteins are distinct, with the Ser135, Cys140 mutant displaying a thiolate-flavin charge-transfer band (Cys140 pKa = 5.2), confirming that Cys140, not Cys135, is in charge-transfer distance both in this mutant and in two electron reduced native enzyme. The native and both mutant proteins are dimers and are precipitated by antibody to native enzyme. Thiol titrations with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) indicate that both mutants contain three kinetically accessible thiols in both oxidized and reduced states. The native enzyme has two titratable thiols when oxidized and four in the two electron reduced state. The native and two Cys to Ser mutant enzymes show differentiable NADPH-dependent catalytic behavior with Hg(SR)2 (R = CH2CH2OH), Hg(CN)2, DTNB, thio-NADP+, and O2, the most striking of which are the activities toward the Hg(II) complexes and DTNB. Only native enzyme reduces Hg(SR)2. The Ser135, Cys140 enzyme alone shows sustained Hg(CN)2 reduction, whereas the native and Cys135, Ser140 enzymes are rapidly inactivated. DTNB reduction is catalyzed by the native and Cys135, Ser140 enzymes, but not by the Ser135, Cys140 enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000936:Antigen-Antibody Complex; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007106:Immune Sera; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010101:Oxygen Consumption; D010957:Plasmids
D000936:Antigen-Antibody Complex; D004220:Disulfides; D007106:Immune Sera; D010088:Oxidoreductases; C021551:mercuric reductase