Redox-mediated transcriptional activation in a CooA variant

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO at a reduced (Fe(II)) heme moiety with resulting conformational changes that promote DNA binding. In this study, we report a variant of CooA, M124R, that is active in transcriptional activation in a redox-dependent manner. Where wild-type CooA is active only in the Fe(II) + CO form, M124R CooA is active in both Fe(II) + CO and Fe(III) forms. Analysis of the pH dependence of the activity of Fe(III) M124R CooA demonstrated that the activity was also coordination state-dependent with a five-coordinate, high-spin species identified as the active form and Cys(75) as the retained ligand. In contrast, the active Fe(II) + CO forms of both wild-type and M124R CooA are six-coordinate and low-spin with a protein ligand other than Cys(75), so that WT and Fe(III) M124R CooA are apparently achieving an active conformation despite two different heme coordination and ligation states. A hypothesis to explain these results is proposed. This study demonstrates the utility of CooA as a model system for the isolation of functionally interesting heme proteins.