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 conformati onal changes that promote DNA binding, In this study, we report a variant o f CooA, M124R, that is active in transcriptional activation in a redox-depe ndent manner. Where wild-type CooA is active only in the Fe-II + CO form, M 124R 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 acti vity was also coordination state-dependent with a five-coordinate, high-spi n 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), s o that WT and Fe-III M124R CooA are apparently achieving an active conforma tion despite two different heme coordination and ligation states. A hypothe sis to explain these results is proposed. This study demonstrates the utili ty of CooA as a model system for the isolation of functionally interesting heme proteins.