Identification of two important heme site residues (cysteine 75 and histidine 77) in CooA, the CO-sensing transcription factor of Rhodospirillum rubrum

The GO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (Fe-III) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys(75) is a heme Ligand in Fe-III CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys(75) is not a ligand in Fe-II CooA. Mutational analysis of all CooA His residues showed that His(77) is critical for GO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its Fen electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His(77) appears to be an axial ligand to Fe-II CooA. These results imply a ligand switch from Cys(75) to His(77) upon reduction of CooA. In addition, an interaction has been identified between Cys(75) and His(77) in Fe-III CooA that may be involved in the GO-sensing mechanism. Finally, His(77) is necessary for the proper conformational change of CooA upon CO binding.