Binding of CO at the Pro² side is crucial for the activation of CO-sensing transcriptional activator CooA. ¹H NMR spectroscopic studies

CooA is a heme-containing transcriptional activator that anaerobically binds to DNA at CO atmosphere. To obtain information on the conformational transition of CooA induced by CO binding to the heme, we assigned ring current-shifted ¹H NMR signals of CooA using two mutants whose axial ligands of the heme were replaced. In the absence of CO, the NMR spectral pattern of H77Y CooA, in which the axial histidine (His⁷⁷) was replaced with tyrosine, was similar to that of wild-type CooA. In contrast, the spectra of CooAΔN5, in which the NH₂ termini including the other axial ligand (Pro²) were deleted, were drastically modulated. We assigned three signals of wild-type CooA at -4.5, -3.6, and -2.8 ppm to δ₁-, α-, and δ₂-protons of Pro ², respectively. The Pro² signals were undetectable in the upfield region of the spectrum of the CO-bound state, which confirms that CO displaces Pro². Interestingly, the Pro² signals were observed for CO-bound H77Y CooA, implying that CO binds to the trans position of Pro² in H77Y CooA. The abolished CO-dependent transcriptional activity of H77Y CooA is therefore the consequence of Pro² ligation. These observations are consistent with the view that the movement of the NH ₂ terminus triggers the conformational transition to the DNA binding form.