ASSIGNMENT OF THE HYPERFINE-SHIFTED H-1-NMR SIGNALS OF THE HEME IN THE OXYGEN SENSOR FIXL FROM RHIZOBIUM-MELILOTI

Background: The Rhizobial oxygen sensor FixL is a hemoprotein with kin ase activity. On binding of strong-field ligands, a change of the ferr ous or ferric hems iron from high to low spin reversibly inactivates t he kinase. This spin-state change and other information on the heme po cket have been inferred from enzymatic assays, absorption spectra and mutagenesis studies. We set out to investigate the spin-state of the F ixL heme and to identify the hyperfine-shifted heme-proton signals by NMR spectroscopy. Results: Using one-dimensional NMR we directly obser ved the high- and low-spin nature of the met- and cyanomet-fixL. heme domain, respectively. We determined the hyperfine-shifted H-1-NMR sign als of the heme and the proximal histidine by one- and two-dimensional spectroscopy and note the absence of distal histidine signals. Conclu sions: These findings support the spin-state mechanism of FixL regulat ion. They establish that the site of heme coordination is a histidine residue and strongly suggest that a distal histidine is absent. With a majority of the heme resonances identified, one- and two-dimensional NMR techniques can be extended to provide structural and mechanistic i nformation about the residues that line the heme pocket.