(Invited) Molecular Mechanisms of Signal Transduction in Heme-Containing Oxygen Sensor Proteins

In heme-based gas sensor proteins, heme acts as the sensing site for binding of gaseous molecules, including O 2 , NO and CO, and indirectly regulates many physiological functions, including the activities of protein kinases, guanylate cyclase, phosphodiesterase, and transcriptional regulatory factors, in response to gas availability. Conceptually, these proteins are always composed of at least two domains: one is a sensor domain (heme-based gas sensing) and the other is a functional domain. However, the structure-function relationship and mechanisms of communication between these domains have not been fully understood. Therefore, we selected a model system, namely a globin-coupled histidine kinase, Af GcHK, in order to study the signal transduction in heme-containing oxygen sensor proteins. The Af GcHK is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Once the oxygen molecule (as the first signal) binds to the heme iron complex in the sensor domain of Af GcHK, the functional domain is stimulated, leading to autophosphorylation at a conserved His residue in the functional domain. The phosphate group of phosphorylated Af GcHK is then transferred to the cognate response regulator. Several biochemical approaches were utilized in order to study the signal transduction between the sensor and function domains of Af GcHK: (i) enzyme kinetic study, (ii) hydrogen/deuterium exchange experiments associated with mass spectrometry, (iii) cross-linking studies and (iv) small-angle X-ray scattering technique. Overall our results indicated that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme’s catalytic activity of the function domain because they modulate its ATP binding affinity and thus change its k cat / K m ATP value. The possible contact area between sensor and function domains was reveled by hydrogen/deuterium exchange experiments associated with mass spectrometry and cross-linking studies. Small-angle X-ray scattering results offered low-resolution model of the particular domain orientation in solution. All these data together will be discussed in order to illustrate the mechanism of signal transduction in the globin-coupled histidine kinase as a representative of heme-containing oxygen sensor proteins. References: Kitanishi K., Kobayashi K., Uchida T., Ishimori K., Igarashi J., Shimizu T.: Identification and functional and spectral characterization of a