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 activates the transcription of genes required for the anaerobic oxidation of CO to CO(2) 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 Fe(II) form of CooA allows high-affinity binding of cyanide (K(d) approximately 0.4 mM) to the heme. In contrast, neither these same variants in the Fe(III) form nor wild-type CooA in either oxidation state was able to bind cyanide even at high concentrations (50 mM). Examination of the pH dependence of spectral changes upon addition of cyanide suggested that the cyanide anion coordinated the heme iron. In addition, the UV-visible absorption spectrum of H77Y Fe(II) CooA without added effectors is also pH-dependent, suggesting that an ionizable amino acid has become solvent-accessible in the absence of His(77). 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.
Thorsteinsson
M V
MV
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Kerby
R L
RL
Roberts
G P
GP
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism*; D003486:Cyanides / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D036781:Fimbriae Proteins; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000378:metabolism; D015533:Transcriptional Activation
D001426:Bacterial Proteins; C088904:CooA protein, E coli; D003486:Cyanides; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D011994:Recombinant Proteins; D036781:Fimbriae Proteins; D006639:Histidine; D002248:Carbon Monoxide