pH dependence of cyanide and imidazole binding to the heme domains of Sinorhizobium meliloti and Bradyrhizobium japonicum 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 pH5 and 11. KD determinations were made at integral pH values, with the strongest binding at pH9 for both ligands. KD for the cyanide complexes of BjFixLH and SmFixLH is 0.15±0.09 and 0.50±0.20μM, respectively, and 0.70±0.01mM for imido-BjFixLH. The association rate constants are pH dependent with maximum values of 443±8 and 252±61M(-1)s(-1) for cyano complexes of BjFixLH and SmFixLH and (5.0±0.3)×10(4) and (7.0±1.4)×10(4)M(-1)s(-1) for the imidazole complexes. The dissociation rate constants are essentially independent of pH above pH5; (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 1270s(-1) for BjFixLH and SmFixLH, respectively.
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, United States of America.
Anil K
AK
Bidwai
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, United States of America.
Angela J
AJ
Ahrendt
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, United States of America.
John S
JS
Sullivan
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, United States of America.
Lidia B
LB
Vitello
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, United States of America. Electronic address: jerman@niu.edu.
James E
JE
Erman
D001426:Bacterial Proteins / Q000737:chemistry*; D020369:Bradyrhizobium / Q000378:metabolism*; D003486:Cyanides / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry*; D000071677:Histidine Kinase; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D009593:Nitroimidazoles / Q000737:chemistry*; D017354:Point Mutation; D011485:Protein Binding; D000072417:Protein Domains; D016962:Sinorhizobium meliloti / Q000378:metabolism*
D001426:Bacterial Proteins; D003486:Cyanides; D006420:Hemeproteins; D008024:Ligands; D009593:Nitroimidazoles; D006418:Heme; D007501:Iron; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; C052587:4-nitroimidazole
Cyanide binding; Equilibrium dissociation constant; FixL; Heme domains; Imidazole binding; Kinetics