The essential catalytic redox couple in arsenate reductase from Staphylococcus aureus.
Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite), which is subsequently extruded from the cell. ArsC couples to thioredoxin, thioredoxin reductase, and NADPH to be enzymatically active. A novel purification method leads to high production levels of highly pure enzyme. A reverse phase method was introduced to systematically analyze and control the oxidation status of the enzyme. The essential cysteinyl residues and redox couple in arsenate reductase were identified by a combination of site-specific mutagenesis and endoprotease-digest mass spectroscopy analysis. The secondary structures, as determined with CD, of wild-type ArsC and its Cys mutants showed a relatively high helical content, independent of the redox status. Mutation of Cys 10, 82, and 89 led to redox-inactive enzymes. ArsC was oxidized in a single catalytic cycle and subsequently digested with endoproteinases ArgC, AspN, and GluC. From the peptide-mass profiles, cysteines 82 and 89 were identified as the redox couple of ArsC necessary to reduce arsenate to arsenite.
Messens
J
J
Dienst Ultrastructuur, Vlaams Interuniversitair instituut voor Biotechnologie, Vrije Universiteit Brussel, Belgium. jmessens@vub.ac.be
Hayburn
G
G
Desmyter
A
A
Laus
G
G
Wyns
L
L
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D053501:Arsenite Transporting ATPases; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016623:Ion Pumps; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013211:Staphylococcus aureus / Q000201:enzymology*; D013880:Thioredoxin-Disulfide Reductase / Q000096:biosynthesis / Q000235:genetics
D016623:Ion Pumps; D009097:Multienzyme Complexes; D013880:Thioredoxin-Disulfide Reductase; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; D003545:Cysteine