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.
Journal of Inorganic Biochemistry
Bidwai, A. K.,
Ahrendt, A. J.,
Sullivan, J. S.,
Vitello, L. B.,
Erman, J. E.
pH dependence of cyanide and imidazole binding to the heme domains of Sinorhizobium meliloti and Bradyrhizobium japonicum FixL.
Journal of Inorganic Biochemistry,
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