The Influence of pH Variation on CooA Activity

CooA, a CO-sensing heme protein, acts as a transcriptional activator of CO-metabolizing proteins in bacteria such as Rhodospirillum rubrum and Carboxydothermus hydrogenoformans through sequence-specific DNA binding. Previous research indicated a reduced iron center and CO gas were necessary for CooA to achieve its active conformation and bind DNA. To determine if other reaction conditions facilitate CooA activation, the role of pH on CooA function was tested. Specifically, a fluorescence anisotropy assay was employed to measure possible Fe(III) CooA DNA binding from pH 3 - 12. Interestingly, CooA was observed to bind DNA without CO at acidic conditions, with optimal binding observed at pH ~3. These results are discussed in light of the normal CO-dependent activation mechanism of CooA proteins

Acid Induced DNA Binding of CooA

A bacterial heme protein, CooA, changes shape to bind DNA and activate transcription. Previous research indicated gas molecule (e.g. carbon monoxide) binding to CooA is essential to initiate protein shape change, but recent studies suggested the protein may be able to bind DNA without gas in acidic conditions. To investigate this, CooA was subjected to an acid range of 4-10 pH units and DNA binding was measured via a fluorescence anisotropy assay. From these studies, pH dependent DNA binding was observed, and optimal binding was achieved in the pH 4-6 range. Addition of gas did not improve DNA binding at these pH values. Ongoing studies are investigating the molecular basis of this effect

The Influence of pH Variation on CooA Activity

A bacterial heme protein, CooA, changes shape to bind DNA and activate transcription. Previous research indicated gas molecule (e.g. carbon monoxide) binding to CooA is essential to initiate protein shape change, but recent studies suggested the protein may be able to bind DNA without gas in acidic conditions. To investigate this, CooA was subjected to an acid range of 4-10 pH units and DNA binding was measured via a fluorescence anisotropy assay. From these studies, pH dependent DNA binding was observed, and optimal binding was achieved in the pH 4-6 range. Addition of gas did not improve DNA binding at these pH values. Ongoing studies are investigating the molecular basis of this effect

Unexpected pH-dependent DNA Binding of CooA, a CO-Sensing Transcription Factor from Rhodospirillum rubrum

CooA, a CO-sensing heme protein, acts as a transcriptional activator of CO metabolizing proteins in Rhodospirillum rubrum and other bacteria through sequence-specific DNA binding. Prior research has indicated that a reduced iron center and CO gas were necessary for CooA to achieve its active conformation and bind DNA. To determine if other reaction conditions facilitate CooA activation, the role of pH on CooA DNA binding was tested. Using a fluorescence anisotropy assay, Fe(III) CooA was observed to bind DNA without the presence of gas under acidic conditions (pH \u3c 7). Specifically, DNA binding was quantified from pH 4 – 10, and optimal binding was observed at approximately pH 4. These results are discussed in light of the normal CO-dependent activation mechanism of CooA proteins