Interaction of the Full-Length Heme-Based CO Sensor Protein RcoM-2 with Ligands.

The heme-based and CO-responsive RcoM transcriptional regulators from are known to display an extremely high affinity for CO while being insensitive to O. We have quantitatively characterized the heme-CO interaction in full-length RcoM-2 and compared it with the isolated heme domain RcoMH-2 to establish the origin of these characteristics. Whereas the CO binding rates are similar to those of other heme-based sensor proteins, the dissociation rates are two to three orders of magnitude lower. The latter property is tuned by the yield of CO escape from the heme pocket after disruption of the heme-CO bond, as determined by ultrafast spectroscopy. For the full-length protein this yield is ∼0.5%, and for the isolated heme domain it is even lower, associated with correspondingly faster CO rebinding kinetics, leading to values of 4 and 0.25 nM, respectively. These differences imply that the presence of the DNA-binding domain influences the ligand-binding properties of the heme domain, thus abolishing the observed quasi-irreversibility of CO binding to the isolated heme domain. RcoM-2 binds target DNA with high affinity ( < 2 nM) when CO is bound to the heme, and the presence of DNA also influences the heme-CO rebinding kinetics. The functional implications of our findings are discussed.