title: Mutagenesis of the redox-active disulfide in mercuric ion reductase: catalysis by mutant enzymes restricted to flavin redox chemistry.
abstract: Mercuric reductase, a flavoenzyme that possess a redox-active cystine, Cys135Cys140, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, we have constructed mutants lacking a redox-active disulfide by eliminating Cys135 (Ala135Cys140), Cys140 (Cys135Ala140), or both (Ala135Ala140). Additionally, we have made double mutants that lack Cys135 (Ala135Cys139Cys140) or Cys140 (Cys135Cys139Ala140) but introduce a new Cys in place of Gly139 with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH2 redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. These differences are manifested in a 23-nm range in enzyme-bound FAD lambda max values, an 80-nm range in thiolate to flavin charge-transfer absorbance maxima, and a ca. 100-mV range in FAD reduction potential. Preliminary evidence for the Ala135Cys139Cys140 mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala135Cys140 protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. For these activities, there is a linear correlation between log kappa cat and enzyme-bound FAD reduction potential. In a sensitive Hg(II)-mediated enzyme-bound FADH2 reoxidation assay, all mutant enzymes were able to undergo at least one catalytic event at rates 50-1000-fold slower than that of the wild-type enzyme. We have also observed the reduction of Hg(II) by free FADH2. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. We conclude that the Cys135 and Cys140 thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: M D
authors.initials: MD
authors.lastname: Distefano
authors.forename: K G
authors.initials: KG
authors.lastname: Au
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D009249:NADP / Q000378:metabolism; D015345:Oligonucleotide Probes; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D013053:Spectrophotometry
chemical_list: D004220:Disulfides; D015345:Oligonucleotide Probes; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D010088:Oxidoreductases; C021551:mercuric reductase