The essential catalytic redox couple in arsenate reductase from Staphylococcus aureus

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistan ce plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic A s(III) (arsenite), which is subsequently extruded from the cell, ArsC coupl es to thioredoxin, thioredoxin reductase, and NADPH to be enzymatically act ive. A novel purification method leads to high production levels of highly pure enzyme. A reverse phase method was introduced to systematically analyz e and control the oxidation status of the enzyme, The essential cysteinyl r esidues and redox couple in arsenate reductase were identified by a combina tion of site-specific mutagenesis and endoprotease-digest mass spectroscopy analysis. The secondary structures, as determined with CD, of wild-type Ar sC and its Cys mutants showed a relatively high helical content, independen t 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 di gested with endoproteinases ArgC, AspN, and GluC. From the peptide-mass pro files, cysteines 82 and 89 were identified as the redox couple of ArsC nece ssary to reduce arsenate to arsenite.