NONSTERIC FACTORS DOMINATE BINDING OF NITRIC-OXIDE, AZIDE, IMIDAZOLE,CYANIDE, AND FLUORIDE TO THE RHIZOBIAL HEME-BASED OXYGEN SENSOR FIXL

Background: The FixL protein is a heme-based sensor. Binding of oxygen to a unique heme domain inhibits a kinase domain of the type found in two-component regulators. Oxygen association is slow, but the dissoci ation rate is comparable to that of myoglobins. We have probed the siz e and chemistry of the FixL heme pocket by measuring the affinities, o n rates and off rates for a wide variety of ferric heme ligands. Cyani de, but not fluoride, regulates the kinase activity. To examine how th e sensory heme domain interacts with the kinase, we asked how the pres ence of the kinase domain affects ligand binding. Results: The affinit ies of ferric FixL for heme ligands follow the same trend as their pK( a) values: cyanide > 4-methyl imidazole > imidazole > fluoride > azide much greater than thiocyanate. The association rates follow the rever se trend. Striking differences from myoglobin include a 6-fold greater affinity for, and faster binding to, the bulky ligand imidazole, a 14 -fotd faster on rate for nitric oxide, a 2 800-fold lower affinity for azide, and a complete failure to bind thiocyanate. The presence of th e kinase domain does not alter the affinity or binding kinetics of the high-spin ligand fluoride, but affects the off rates of other ligands , The EPR spectrum shows a characteristic pentacoordinate nitrosyl hem e, indicating that the Fe-His bond in FixL is strained. Conclusions: T he importance of ligand deprotonation to the on rates and the fact tha t large ligands bind readily indicate that the heme pocket is open and apolar. Ligand basicity strongly influences the strength of binding. The destabilization of inhibitory ligands by the presence of the kinas e domain is consistent with a 'load' imposed by coupling to the inacti vating mechanism.