Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

Journal Article · Tue Feb 07 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00429a035· OSTI ID:5345855

Distefano, M D; Au, K G; Walsh, C T ^[1]

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1. 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), Journal Name: Biochemistry; (USA) Vol. 28:3; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201 -- Biochemistry-- Tracer Techniques
560300* -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACIDS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHLORIDES
CHLORINE COMPOUNDS
CYSTINE
DAYS LIVING RADIOISOTOPES
DISULFIDES
ENZYMES
ESCHERICHIA COLI
EVAPORATION
EVEN-ODD NUCLEI
HALIDES
HALOGEN COMPOUNDS
HEAVY NUCLEI
ISOTOPES
LABELLED COMPOUNDS
LIGANDS
MERCURY 203
MERCURY CHLORIDES
MERCURY COMPOUNDS
MERCURY HALIDES
MERCURY ISOTOPES
MICROORGANISMS
MUTAGENESIS
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
PHASE TRANSFORMATIONS
RADIOISOTOPES
REDOX REACTIONS
REDUCTION