CooA is a transcriptional regulator that senses carbon monoxide (CO) through a heme moiety and activates the transcription of the coo regulon, whose gene products are required for the oxidation of CO in Rhodospirillium rubrum. Preliminary work suggests a low-spin, hexacoordinate heme structure in the ferric, ferrous, and CO-ferrous forms of CooA, suggesting that a protein ligand is displaced upon CO binding. The precise mechanism of signal transduction within CooA upon CO binding is currently unknown. In order to address this question, the proposal describes the creation and characterization of functionally important variants of CooA that show an altered response to CO and the potential effectors nitric oxide and cyanide, is proposed. Functionally interesting CooA variants will be isolated and characterized by standard spectroscopies in order to interpret the effects of perturbation of the proposed heme-binding pocket of CooA. The study proposed will produce information concerning the interpretation of the CO signal by CooA and how this event is coupled to DNA binding by conformational changes within the protein. Characterization f mutants that demonstrate atypical responses to effectors will provide an understanding of CooA function and insights concerning small molecule sensing and discrimination by proteins.
