Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 36, Pages 26382-26390Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M602158200
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- NCRR NIH HHS [1P20RR17675] Funding Source: Medline
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Desulfitobacterium dehalogenans can use chlorinated aromatics including polychlorinated biphenyls as electron acceptors in a process called dehalorespiration. Expression of the cpr gene cluster involved in this process is regulated by CprK, which is a member of the CRP/FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. High affinity interaction of the chlorinated aromatic compound with the effector domain of CprK triggers binding of CprK to an upstream target DNA sequence, which leads to transcriptional activation of the cpr gene cluster. When incubated with oxygen or diamide, CprK undergoes inactivation; subsequent treatment with dithiothreitol restores activity. Using mass spectrometry, this study identifies two classes of redox-active thiol groups that form disulfide bonds upon oxidation. Under oxidative conditions, Cys(105), which is conserved in FNR and most other CprK homologs, forms an intramolecular disulfide bond with Cys(111), whereas an intermolecular disulfide bond is formed between Cys(11) and Cys(200). SDS-PAGE and site-directed mutagenesis experiments indicate that the Cys(11)/Cys(200) disulfide bond links two CprK subunits in an inactive dimer. Isothermal calorimetry and intrinsic fluorescence quenching studies show that oxidation does not change the affinity of CprK for the effector. Therefore, reversible redox inactivation is manifested at the level of DNA binding. Our studies reveal a strategy for limiting expression of a redox-sensitive pathway by using a thiol-based redox switch in the transcription factor.
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