Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by an operon of three genes in which arsC codes for an arsenate reductase with unique characteristics. Here we describe the identification of two additional and nearly identical genes coding for arsenate reductases in Synechocystis sp. strain PCC 6803, which we have designed arsI1 and arsI2, and the biochemical characterization of both ArsC (arsenate reductase) and ArsI. Functional analysis of single, double, and triple mutants shows that both ArsI enzymes are active arsenate reductases but that their roles in arsenate resistance are essential only in the absence of ArsC. Based on its biochemical properties, ArsC belongs to a family that, though related to thioredoxin-dependent arsenate reductases, uses the glutathione/glutaredoxin system for reduction, whereas ArsI belongs to the previously known glutaredoxin-dependent family. We have also analyzed the role in arsenate resistance of the three glutaredoxins present in Synechocystis sp. strain PCC 6803 both in vitro and in vivo. Only the dithiolic glutaredoxins, GrxA (glutaredoxin A) and GrxB (glutaredoxin B), are able to donate electrons to both types of reductases in vitro, while GrxC (glutaredoxin C), a monothiolic glutaredoxin, is unable to donate electrons to either type. Analysis of glutaredoxin mutant strains revealed that only those lacking the grxA gene have impaired arsenic resistance.Arsenic is a ubiquitous pollutant that can produce cancer and cause serious health problems in certain parts of the world (36). Because of the wide distribution of arsenic compounds, arsenic resistance is widespread among living organisms (47). Most resistance systems reduce arsenate to arsenite and export the latter to the outside of the cell or transport it to a vacuole (31). Arsenate reduction to arsenite is catalyzed by arsenate reductase, an enzyme at least three families of which have been described (25,30,43). The first family is exemplified by Escherichia coli ArsC, a protein that uses the glutathione (GSH)/ glutaredoxin system as a reducing system and has a single catalytic cysteine. The second family is represented by Staphylococcus aureus and Bacillus subtilis arsenate reductases (also named ArsC). These enzymes are related to low-molecularweight protein phosphotyrosine phosphatases and use thioredoxin as a reducing system through an intramolecular redox cascade requiring three cysteines for arsenate reduction (25). The last family is present only in eukaryotic organisms and was initially described in Saccharomyces cerevisae and, more recently in Leishmania major and Arabidopsis thaliana (6,32,53). These arsenate reductases also use the GSH/glutaredoxin system as a reductant, but they are related not to the E. coli reductase but rather to the Cdc25 family of protein phosphatases.Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by an operon of three genes that is regulated by an unlinked arsR homolog. The operon includes an arsenite transporter gene, arsB; an arsH homolog witho...