Altering the specificity of CooA, the carbon monoxide-sensing transcriptional activator: Characterization of CooA variants that bind cyanide in the Fe-II form with high affinity

CooA is a carbon monoxide- (CO-) sensing homodimeric heme protein that acti vates the transcription of genes required for the anaerobic oxidation of CO to CO2 in the phototrophic bacterium Rhodospirillum rubrum. In this study, we demonstrate that mutational alteration of the histidine residue (His(77 )) that serves as a heme ligand in the Felt form of CooA allows high-affini ty binding of cyanide (Kd similar to 0.4 mM) to the heme. In contrast, neit her these same variants in the Fe-III form nor wild-type CooA in either oxi dation state was able to bind cyanide even at high concentrations (50 mM). Examination of the pH dependence of spectral changes upon addition of cyani de suggested that the cyanide anion coordinated the heme iron. In addition, the UV-visible absorption spectrum of H77Y Fe-II CooA without added effect ors is also pH-dependent, suggesting that an ionizable amino acid has becom e solvent-accessible in the absence of His77. Finally, we demonstrate that the transcriptional activity of H77Y CooA shows a small (1.4-fold) increase in the presence of cyanide, suggesting that the binding of cyanide to this variant promotes the active conformation of H77Y CooA.