Binding of CO at the Pro(2) side is crucial for the activation of CO-sensing transcriptional activator CooA - H-1 NMR spectroscopic studies

CooA is a heme-containing transcriptional activator that anaerobically bind s to DNA at CO atmosphere. To obtain information on the conformational tran sition of CooA induced by CO binding to the heme, we assigned ring current- shifted H-1 NMR signals of CooA using two mutants whose axial Ligands of th e heme were replaced. In the absence of CO, the NMR spectral pattern of H77 T CooA in which the axial histidine (His(77)) was replaced with tyrosine, w as similar to that of wild-type CooA. In contrast, the spectra of CooA Delt a N5, in which the NH2 termini including the other axial ligand (Pro(2)) we re deleted, were drastically modulated. We assigned three signals of wild-t ype CooA at -4.5, -3.6, and -2.8 ppm to delta (1)-, alpha-, and delta (2)-p rotons of Pro(2), respectively. The Pro(2) signals were undetectable in the upfield region of the spectrum of the CO-bound state, which confirms that CO displaces Pro(2), Interestingly, the Pro(2) signals were observed for CO -bound H77Y CooA implying that CO binds to the trans position of Pro(2) in H77Y CooA. The abolished CO-dependent transcriptional activity of H77Y CooA is therefore the consequence of Pro(2) ligation. These observations are co nsistent with the view that the movement of the NH2 terminus triggers the c onformational transition to the DNA binding form.