XAS STUDIES OF NOVEL ARSENIC BINDING SITES IN AS (III)-RESPONSIVE TRANSCRIPTIONA

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this project is characterization of arsenic binding sites in proteins involved in arsenic detoxification. ArsRs are As(III)-responsive transcriptional repressors that regulate expression of arsenic detoxification genes. We have identified three homologous ArsRs from three different bacteria but apparently have evolved different arsenic binding sites in different locations in each protein. The first is the well-characterized ArsR from plasmid R773. The binding site in this repressor is an S3 site composed of three co-linear cysteine residues, Cys32, Cys34 and Cys37 in the DNA binding domain. The second is an ArsR from Acidothiobacillus ferrooxidans, a sulfuric acid-producing bacterium used in the gold mining industry. Preliminary EXAFS results suggest that the site in this ArsR is a mixed S/O/N site, suggesting an S2 site and a hydroxyl ligand. This repressor has a C-terminal vicinal cysteine pair residues that may form an As(III) binding site. The third ArsR is from In Corynebacterium glutamicum, which is used for the production of glutamic acid. This ArsR has an N-terminal vicinal cysteine pair and a third cysteine near the DNA binding domain that we hypothesize forms an S3 site for As(III). EXAFS of each of the three repressors with bound As(III) will differentiate between these possibilities. Mutant ArsRs lacking cysteine residues, singly and in combination, will be used to identify the specific ligands in each site. ArsD is a novel As(III) metallochaperone. It has three vicinal cysteine pairs and several other cysteines. Cys12,13 and 18 have been identified by mutagenesis as required for chaperone activity. ArsD mutants in these cysteine residues, singly and in combination, will be used to characterize the As(III) binding site. ArsM is a newly identified As(III)-SAM methyltransferase that methylates As(III) to a variety of species. Participation of conserved residues Cys30 and 31 in As(III) binding site will be investigated.