Bidwai Anil K, Ahrendt Angela J, Sullivan John S, Vitello Lidia B, Erman James E
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, United States of America.
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, United States of America.
J Inorg Biochem. 2015 Dec;153:88-102. doi: 10.1016/j.jinorgbio.2015.10.003. Epub 2015 Oct 22.
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.
已研究了氰化物和咪唑在pH5至11范围内与苜蓿中华根瘤菌和日本慢生根瘤菌FixL的血红素结构域（SmFixLH和BjFixLH）结合的平衡和动力学性质。在整数pH值下测定解离常数（KD），两种配体在pH9时结合最强。BjFixLH和SmFixLH的氰化物配合物的KD分别为0.15±0.09和0.50±0.20μM，亚氨基-BjFixLH的KD为0.70±0.01mM。缔合速率常数与pH有关，BjFixLH和SmFixLH的氰化物配合物的最大值分别为443±8和252±61M⁻¹s⁻¹，咪唑配合物的为(5.0±0.3)×10⁴和(7.0±1.4)×10⁴M⁻¹s⁻¹。解离速率常数在pH5以上基本与pH无关；BjFixLH和SmFixLH氰化物配合物的解离速率常数分别为(1.2±0.3)×10⁻⁴和(1.7±0.3)×10⁻⁴s⁻¹，咪唑配合物的为(73±19)和(77±14)s⁻¹。FixLH中的两个可电离基团影响配体结合速率。酸性较强的基团被鉴定为血红素6丙酸，在BjFixLH中的pKa为7.6±0.2，在SmFixLH中的pKa为6.8±0.2。第二次电离是由于形成了羟基-FixLH，BjFixLH的pKa值为9.64±0.05，SmFixLH的pKa值为9.61±0.05。咪唑结合受血红素口袋打开速率的限制，BjFixLH和SmFixLH观察到的最大值分别为680和1270s⁻¹。
