Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry
- Harvard Medical School, Boston, MA (USA)
Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys{sub 135}Cys{sub 140}, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys{sub 135} (Ala{sub 135}Cys{sub 140}), Cys{sub 14} (Cys{sub 135}Ala{sub 140}), or both (Ala{sub 135}Ala{sub 140}). Additionally, they have made double mutants that lack Cys{sub 135} (Ala{sub 135}Cys{sub 139}Cys{sub 140}) or Cys{sub 140} (Cys{sub 135}Cys{sub 139}Ala{sub 140}) but introduce a new Cys in place of Gly{sub 139} 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/FADH{sub 2} redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala{sub 135}Cys{sub 139}Cys{sub 14} 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 Ala{sub 135}Cys{sub 140} 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. 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. They conclude that the Cys{sub 135} and Cys{sub 140} thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.
- OSTI ID:
- 5345855
- Journal Information:
- Biochemistry; (USA), Vol. 28:3; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ESCHERICHIA COLI
REDOX REACTIONS
LABELLED COMPOUNDS
EVAPORATION
MERCURY CHLORIDES
REDUCTION
OXIDOREDUCTASES
MUTAGENESIS
BIOLOGICAL EFFECTS
CYSTINE
LIGANDS
MERCURY 203
AMINO ACIDS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHLORIDES
CHLORINE COMPOUNDS
DAYS LIVING RADIOISOTOPES
DISULFIDES
ENZYMES
EVEN-ODD NUCLEI
HALIDES
HALOGEN COMPOUNDS
HEAVY NUCLEI
ISOTOPES
MERCURY COMPOUNDS
MERCURY HALIDES
MERCURY ISOTOPES
MICROORGANISMS
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHASE TRANSFORMATIONS
RADIOISOTOPES
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