CooA of Rhodospirillum rubruin is a CO-sensing, heme-containing transcriptional activator that regulates the expression of the genes responsible for CO oxidation. We randomized the codons for residues 75-77 of CooA which include two proximal heme ligands, screened for both CO-dependent and CO-independent variants, and characterized in vivo and in vitro properties of selected CooA variants. The analysis showed that small residues at position 75 are critical and that, as previously suspected, His77 is absolutely necessary for CO responsiveness of CooA. Many hemetess variants altered at those residues were found to be constitutively active. We propose that proximal heme pocket residues of wild-type CooA have important role in stabilizing both active and inactive heme positions for its CO-sensing function. (c) 2006 Elsevier Inc. All rights reserved.