Mechanistic Studies of the Carbon Monoxide Sensor CooA

CooA is a protein found in Rhodospirillum rubrum (Rr) and Carboxydothermus hydrogenoformans (Ch) and is both a carbon monoxide (CO) sensor and transcription factor. When CO binds to a CooA iron group (called heme), the CooA protein undergoes a shape change that enables it to bind to DNA. The broader goal of this research is to obtain a mechanistic understanding of how the CooA heme iron, along with amino acids that are attached to that iron, enable CooA to act as a CO-specific sensor. In this project, experiments that vary both the identity of the gas and the chemicals used to reduce the iron have been performed for both Rr and Ch CooA to study how differences between the amino acids bonded to iron in these two proteins affect gas sensing. Using UV-Visible spectroscopy to study the CooA iron bonding environment and fluorescence spectroscopy to study CooA DNA bonding, preliminary data suggest that the order in which heme iron, reducing agent, and CO are added to CooA is significant to the effectiveness of DNA binding. Additionally, reaction kinetics appear to play a crucial role in the binding of gases, DNA, and amino acid side chains to the protein. Current investigations to quantify the impact of the order of chemical addition and reaction kinetics are ongoing. Ultimately, results from these studies may contribute to the development of future therapeutics for iron proteins related to CooA that are found in the human body. Keywords: CooA, heme protein, CO sensor, DNA binding, transcription facto