pH dependence of cyanide and imidazole binding to the heme domains of \u3cem\u3eSinorhizobium meliloti\u3c/em\u3e and \u3cem\u3eBradyrhizobium japonicum\u3c/em\u3e FixL
Equilibrium and kinetic properties of cyanide and imidazole binding to the heme domains of Sinorhizobium meliloti and Bradyrhizobium japonicum FixL (SmFixLH and BjFixLH) have been investigated between pH 5 and 11. KD determinations were made at integral pH values, with the strongest binding at pH 9 for both ligands. KD for the cyanide complexes of BjFixLH and SmFixLH is 0.15 ± 0.09 and 0.50 ± 0.20 μM, respectively, and 0.70 ± 0.01 mM for imido-BjFixLH. The association rate constants are pH dependent with maximum values of 443 ± 8 and 252 ± 61 M−1 s−1 for cyano complexes of BjFixLH and SmFixLH and (5.0 ± 0.3) × 104 and (7.0±1.4) × 104M−1 s−1 for the imidazole complexes. The dissociation rate constants are essentially independent of pH above pH 5; (1.2 ± 0.3) × 10−4 and (1.7 ± 0.3) × 10−4 s−1 for the cyano complexes of BjFixLH and SmFixLH, and (73±19) and (77±14) s−1 for the imidazole complexes. Two ionizable groups in FixLH affect the rate of ligand binding. The more acidic group, identified as the heme 6 propionic acid, has a pKa of 7.6 ± 0.2 in BjFixLH and 6.8 ± 0.2 in SmFixLH. The second ionization is due to formation of hydroxy-FixLH with pKa values of 9.64± 0.05 for BjFixLH and 9.61 ± 0.05 for SmFixLH. Imidazole binding is limited by the rate of heme pocket opening with maximum observed values of 680 and 1270 s−1 for BjFixLH and SmFixLH, respectively
