PDBsum entry 1s3c

Oxidoreductase

PDB id: 1s3c

Contents

Protein chain

138 a.a.

Ligands

SO4 ×3

Metals

_CS ×3

Waters ×276

References listed in PDB file

Key reference

Title

Arginine 60 in the arsc arsenate reductase of e. Coli plasmid r773 determines the chemical nature of the bound as(III) product.

Authors

S.Demel, J.Shi, P.Martin, B.P.Rosen, B.F.Edwards.

Ref.

Protein Sci, 2004, 13, 2330-2340. [DOI no: 10.1110/ps.04787204]

PubMed id

15295115

Abstract

Arsenic is a ubiquitous environmental toxic metal. Consequently, organisms detoxify arsenate by reduction to arsenite, which is then excreted or sequestered. The ArsC arsenate reductase from Escherichia coli plasmid R773, the best characterized arsenic-modifying enzyme, has a catalytic cysteine, Cys 12, in the active site, surrounded by an arginine triad composed of Arg 60, Arg 94, and Arg 107. During the reaction cycle, the native enzyme forms a unique monohydroxyl Cys 12-thiol-arsenite adduct that contains a positive charge on the arsenic. We hypothesized previously that this unstable intermediate allows for rapid dissociation of the product arsenite. In this study, the role of Arg 60 in product formation was evaluated by mutagenesis. A total of eight new structures of ArsC were determined at resolutions between 1.3 A and 1.8 A, with R(free) values between 0.18 and 0.25. The crystal structures of R60K and R60A ArsC equilibrated with the product arsenite revealed a covalently bound Cys 12-thiol-dihydroxyarsenite without a charge on the arsenic atom. We propose that this intermediate is more stable than the monohydroxyarsenite intermediate of the native enzyme, resulting in slow release of product and, consequently, loss of activity.

Figure 4.
Figure 4. Interactions of the Csr12 arsonocysteine adducts. Csr12 and its adjacent residues within 4.0 Å are shown for (A) native ArsC and (B) the R60K mutant. The hydrogen bonds are depicted with dotted lines. The cyan atom in Csr12 is arsenic.

Figure 5.
Figure 5. Reaction mechanism of the R733 ArsC arsenate reductase. The mechanism is consistent with the crystal structures described in Table 1 Go-. In step 1, the free enzyme (structure I) forms the observed covalent intermediate with arsenate (Martin et al. 2001). In step 2, this intermediate is glutathionylated, a structure that has not yet been obtained. In step 3, As(V) is reduced to As(III), producing a dihydroxy arsenite intermediate (structures VI, IX). In step 4, the novel monohydroxy intermediate with a positively charged arsenic is formed (Martin et al. 2001). Finally, in step 5, the free enzyme is regenerated (structure I).

The above figures are reprinted by permission from the Protein Society: Protein Sci (2004, 13, 2330-2340) copyright 2004.