Dual roles of an essential cysteine residue in activity of a redox-regulated bacterial transcriptional activator

CprK from Desulfitobacterium dehalogenans is the first characterized transcriptional regulator of anaerobic dehalorespiration and is controlled at two levels: redox and effector binding. In the reduced state and in the presence of chlorinated aromatic compounds, CprK positively regulates expression of the cpr gene cluster. One of the products of the cpr gene cluster is CprA, which catalyzes the reductive dehalogenation of chlorinated aromatic compounds. Redox regulation of CprK occurs through a thiol/ disulfide redox switch, which includes two classes of cysteine residues. Under oxidizing conditions, Cys(11) and Cys(200) form an intermolecular disulfide bond, whereas Cys(105) and Cys(111) form an intramolecular disulfide. Here, we report that Cys(11) is involved in redox inactivation in vivo. Upon replacement of Cys(11) with serine, alanine, or aspartate, CprK loses its DNA binding activity. C11A is unstable; however, circular dichroism studies demonstrate that the stability and overall secondary structures of CprK and the C11S and C11D variants are similar. Furthermore, effector binding remains intact in the C11S and C11D variants. However, fluorescence spectroscopic results reveal that the tertiary structures of the C11S and C11D variants differ from that of the wild type protein. Thus, Cys(11) plays a dual role as a redox switch and in maintaining the correct tertiary structure that promotes DNA binding.