9724048 Gilles-Gonzalez The rhizobial oxygen sensing protein FixL contains a catalytic domain and a heme binding domain. Apo-FixL or FixL having high-spin heme iron autophosphorylates at a conserved histidine with the (-phosphoryl group from ATP. By contrast, FixL having low-spin heme iron, is enzymatically inactive. The proteins to be exploited in the proposed studies include the full-length FixL from Bradyrhizobium japonicum (BjFixL), a Rhizobium meliloti FixL lacking an ATP binding region RmFixLT(ter439) , the isolated heme domain of the R. meliloti FixL (RmFixLH), and the same domain coupled to kinase (RmFixLT). To probe the nature of the FixL heme pocket and the coupling of the kinase to the heme, a number of factors will be examined that are expected to influence binding of heme ligands. Those factors include: the chemical nature of the heme ligands, the presence of ATP or a phosphoryl group, and the presence of a kinase domain. The kinetic and thermodynamic parameters for binding of F-, CN-, N3-, imidazole, and NO to FeIIIBjFixL will provide information about the influence of ligand shape, size, polarity and charge on binding to the heme iron. Measurements of oxygen binding to FixL complexed with non-hydrolyzable ATP analogs or to FixL lacking a site of ATP binding will extend knowledge of the "back effect" of the kinase. Determination of the activation energies and the enthalpies for binding of ligands of varying crystal-field strengths to RmFixLH and RmFixLT will help to examine the influence of the kinase. Resonance Raman spectroscopy experiments verifying the heme coordination and examining the impact of the kinase on the spin equilibria will complement the measurements of ligand binding. Structural differences in the heme pockets of the cyanomet derivatives of the FixLs will be probed by NMR spectroscopy on isotopically labeled proteins or proteins reconstituted with isotopically labeled heme. More detailed structural information will be obtained from X-ray crystallogra phic studies of high-and low-spin derivatives of FixL heme domains of varying lengths and the full-length BjFixL. The overall goal of this work is to understand the biophysics of signal transduction. It has particular relevance to signal transduction by heme-based sensors such as guanylyl cyclase and by the histidine kinases of the two-component class. An understanding of FixL's regulatory mechanism will provide a key part of a complete biochemical description of the regulation of nitrogen fixation. Possible applications of this research include the design of novel sensors of heme ligands for biotechnology and the development of improved rhizobial strains for agriculture. ***
