Pathways of arsenic uptake and efflux

Abstract

Arsenic is the most prevalent environmental toxic substance and ranks first on the U.S. Environmental Protection Agency's Superfund List. Arsenic is a carcinogen and a causative agent of numerous human diseases. Paradoxically arsenic is used as a chemotherapeutic agent for treatment of acute promyelocytic leukemia. Inorganic arsenic has two biological important oxidation states: As(V) (arsenate) and As(III) (arsenite). Arsenic uptake is adventitious because the arsenate and arsenite are chemically similar to required nutrients. Arsenate resembles phosphate and is a competitive inhibitor of many phosphate-utilizing enzymes. Arsenate is taken up by phosphate transport systems. In contrast, at physiological pH, the form of arsenite is As(OH)(3), which resembles organic molecules such as glycerol. Consequently, arsenite is taken into cells by aquaglyceroporin channels. Arsenic efflux systems are found in nearly every organism and evolved to rid cells of this toxic metalloid. These efflux systems include members of the multidrug resistance protein family and the bacterial exchangers Acr3 and ArsB. ArsB can also be a subunit of the ArsAB As(III)-translocating ATPase, an ATP-driven efflux pump. The ArsD metallochaperone binds cytosolic As(III) and transfers it to the ArsA subunit of the efflux pump. Knowledge of the pathways and transporters for arsenic uptake and efflux is essential for understanding its toxicity and carcinogenicity and for rational design of cancer chemotherapeutic drugs.

Figure 1. Pathways of arsenic uptake and efflux

Arsenate [As(V)] is taken up by phosphate transporters, and As(III) is conducted up by AQPs (GlpF in Escherichia coli, Fps1p in yeast, and AQP7 and AQP9 in mammals). In both E. coli and Saccharomyces cerevisiae, arsenate is reduced to arsenite by arsenate reductases. In E. coli and many other bacteria, arsenite is extruded from the cells by ArsB alone or by the ArsAB ATPase. In many other bacteria, Acr3 replaces ArsB as the plasma membrane arsenite efflux protein. (Some bacteria have both ArsB and Acr3!) Fungi also use Acr3 for arsenite efflux, and yeast Ycf1p, which is a member of the MRP group of the ABC superfamily of drug resistance ATPases, pumps As(GS)₃ into the vacuole. The legume symbiotic bacterium Sinorhizobium meliloti is unique in that it uses an AQP for arsenite efflux rather than uptake. In contrast to most bacteria, S. meliloti is very sensitive to arsenite because it has neither ArsB nor Acr3. It is resistant to arsenate, which, as in other organisms, is brought into cells by the phosphate transporters. The first step of detoxification involves reduction of arsenate to arsenite by ArsC. The AqpS channel facilitates downhill transport of internally generated As(III). Thus, this detoxification mechanism functions to confer arsenate but not arsenite resistance in S. meliloti. See the color plate.

Figure 2. Uptake and efflux of trivalent arsenic species in liver

Inorganic As(OH)₃ flows down its concentration gradient from blood into hepatocytes through AQP9, the liver AQP isoform. In the cytosol of the hepatocyte, As(III) can be either glutathionylated or methylated to MAs(V), which is reduced to MAs(III). As(GS)₃ is pumped into bile by MRP2. Alternatively, As(III) is methylated by the enzyme As(III) SAM methyltransferase (AS3MT) to CH₃As(OH)₂, which then flows down its concentration gradient via AQP9 into blood, to the kidney and eliminated in the urine. See the color plate.

Figure 3. Transfer of As(III) from ArsD to ArsA

The model shows a hypothetical five-step reaction scheme for transfer of As(III) from ArsD to ArsA for extrusion from the cell. Step 1: Intracellular As(OH)₃ complexes with GSH to form As(GS)₃. Step 2: ArsD binds As(III) by exchange of the three thiols of As(GS)₃ for the three thiols of residues Cys12, Cys13 and Cys18. Step 3: The ArsD–As(III) fits into the cavity of the open form of ArsA, with the three thiols of ArsD juxtaposed to the three thiols of the As(III)-binding site of ArsA. Step 4: In a three-step thiol exchange reaction, As(III) is transferred from ArsD to ArsA. Step 5: As(III) binding to ArsA induces a conformational change that increases the rate of ATP hydrolysis and, consequently, the rate of As(III) extrusion by the ArsAB pump. See the color plate.