Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme

Background: In Escherichia coli bearing the plasmid R773, resistance to ars enite, arsenate, antimonite, and tellurite is conferred by the arsRDABC pla smid operon that codes for an ATP-dependent anion pump. The product of the arsC gene, arsenate reductase (ArsC), is required to efficiently catalyze t he reduction of arsenate to arsenite prior to extrusion. Results: Here, we report the first X-ray crystal structures of ArsC at 1.65 Angstrom and of ArsC complexed with arsenate and arsenite at 1.26 Angstrom resolution. The overall fold is unique. The native structure shows sulfate and sulfite ions binding in the active site as analogs of arsenate and ars enite. The covalent adduct of arsenate with Cys-12 in the active site of Ar sC, which was analyzed in a difference map, shows tetrahedral geometry with a sulfur-arsenic distance of 2.18 Angstrom. However, the corresponding add uct with arsenite binds as a hitherto unseen thiarsahydroxy adduct. Finally , the number of bound waters (385) In this highly ordered crystal structure approaches twice the number expected at this resolution for a structure of 138 ordered residues. Conclusions: Structural information from the adduct of ArsC with its substr ate (arsenate) and with its product (arsenite) together with functional inf ormation from mutational and biochemical studies on ArsC suggest a plausibl e mechanism for the reaction. The exceptionally well-defined water structur e indicates that this crystal system has precise long-range order within th e crystal and that the upper limit for the number of bound waters in crysta l structures is underestimated by the structures in the Protein Data Bank.