Impact of Heme Reconstitution on the Spectroscopic Properties and DNA Binding Activity of the H82A Variant of the Carbon Monoxide-Sensing Heme Transcription Factor, CooA
CooA is a gas-sensing heme protein that regulates the expression of genes responsible for growth by carbon monoxide (CO) in several bacteria. CO binding to the CooA heme groups results in an allosteric conformation change that initiates the protein’s DNA binding function. All known CooA homologs have a conserved histidine residue that i) acts as the proximal heme ligand and ii) istrans to CO. Together with the results from mutagenesis studies, this observation suggests the presence of a proximal histidine ligand is required for CooA’s DNA-binding activity. However, since mutants lacking this His have been observed to lose heme upon purification, an alternative hypothesis is that the absence of heme, and not the presence of histidine per se, accounts for this loss of function. To test this hypothesis, we measured the spectroscopic properties and DNA-binding activity of heme-reconstituted H82A. Results obtained using electronic absorbance spectroscopy suggest heme-reconstituted Fe(II)-CO H82A has an iron environment similar to that of wild-type CooA. Preliminary results from a fluorescence anisotropy assay also reveal that reconstituted H82A possesses modest DNA binding activity, which suggests the presence of His-82 is not essential for this activity. The authors wish to thank the Valparaiso University College of Arts and Sciences for financial support.
Troester, Rachel E. and Clark, Robert W., "Impact of Heme Reconstitution on the Spectroscopic Properties and DNA Binding Activity of the H82A Variant of the Carbon Monoxide-Sensing Heme Transcription Factor, CooA" (2010). Chemistry Faculty Publications. Paper 7.