Characterization of variants altered at the N-terminal proline, a novel heme-axial ligand in CooA, the CO-sensing transcriptional activator

Cook the carbon monoxide-sensing transcription factor from Rhodospirillum r ubrum, binds CO through a heme moiety resulting in conformational changes t hat promote DNA binding. The crystal structure shows that the N-terminal Pr o(2) Of one subunit (Met(1) is removed post-translationally provides one li gand to the heme of the other subunit in the Cook homodimer. To determine t he importance of this novel ligand and the contiguous residues to Cook func tion, we have altered the N terminus through two approaches: site directed mutagenesis and regional randomization, and characterized the resulting Coo k variants. While Pro(2) appears to be optimal for Cook function, it is not essential and a variety of studied variants at this position have substant ial CO-sensing function. Surprisingly, even alterations that add a residue (where Pro(2) is replaced by Met(1)-Tyr(2), for example) accumulate heme-co ntaining Cook with functional properties that are similar to those of wild- type Cook Other nearby residues, such as Phe(5) and Asn(6) appear to be imp ortant for either the structural integrity or the function of Cook These re sults are contrasted with those previously reported for alteration of the H is(77) ligand on the opposite side of the heme.