The long-term goal of this project is to understand how CO functions as a cellular signal. There is considerable evidence supporting a role for CO as a regulator in human vascular and neural tissue, but the mechanism of action of CO is as yet incompletely understood. This project is to investigate the only known CO sensor, the hemoprotein CooA from the photosynthetic bacterium Rhodospirillum rubrum, in order to elucidate the mechanism by which this protein senses CO. The presumed receptor for CO in mammalian tissue, soluble guanylyl cyclase, is also a hemoprotein and a number of other hemoproteins are now known to serve as gas sensors. Understanding the function of the CO sensor CooA will provide a foundation for understanding hemoprotein sensors in human physiology. CooA is a member of the CRP (cAMP receptor protein) family of transcription regulators. In the presence of CO, CooA binds to DNA and turns on the expression of a multicomponent CO-oxidation system in R. rubrum. The cooperative binding of oxygen to hemoglobin is the paradigm for understanding how heme can function as a regulator of protein function. Like hemoglobin, CooA is proposed to undergo a conformational change that is triggered by the binding of CO to the heme cofactor. The overall objective of the studies outlined in this proposal is to correlate changes in the heme coordination state of CooA with changes in protein conformation in order to understand how the heme serves to regulate protein function. It is our hypothesis that changes in the coordination environment of the heme upon CO binding alter the conformation of CooA, enabling the protein to bind to DNA. There are three Specific Aims that we propose to address in this project period: 1) we will identify the unique coordination characteristics of the N- terminal proline ligand, 2) we will correlate CO binding and oxidation state with changes in protein conformation, and 3) we will identify the changes in heme coordination that induce DNA binding activity. Control of protein conformation through changes in metal coordination geometry is a general mechanism for gas sensing, and it is likely that many proteins that employ this mechanism remain to be discovered. Understanding the mechanism by which CooA senses CO will add to our knowledge of how heme is used as a gas sensor in biology and suggest mechanisms by which CO functions as a signal in human tissues.
