Jacobson Timothy, Williamson Joy, Wasilewski Anthony, Felesik Janet, Vitello Lidia B, Erman James E
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
Arch Biochem Biophys. 2004 Feb 15;422(2):125-36. doi: 10.1016/j.abb.2003.12.016.
Yeast cytochrome c peroxidase (CcP) and horse metmyoglobin (Mb) bind HN3 with similar affinities at 25 degrees C. The pH-independent equilibrium association constants for formation of the CcP.HN3 and Mb.HN3 complexes are (1.05 +/- 0.06)x10(5) and (1.6 +/- 0.8)x10(5) M(-1), respectively. However, the thermodynamic parameters for formation of the two complexes are quite different. The DeltaH0 values for formation of CcP.HN3 and Mb.HN3 are -16.4 +/- 0.7 and -9.0 +/- 0.5 kcal/mol, respectively, and the Delta S0 values are -32 +/- 2 and -16 +/- 2 cal/deg mol, respectively. The proton associated with HN3 is retained in both protein complexes at low pH but dissociates with apparent pKA values of 5.5 +/- 0.2 and > or =8.2 for the Mb.HN3 and CcP.HN3 complexes, respectively. CcP and Mb differ significantly in their reactivity toward the azide anion, N3-. CcP binds N3- very weakly, if at all, and only an upper-limit of 18 +/-5 M(-1) for the pH-independent equilibrium association constant for the CcP.N3- complex can be determined. Mb binds N3- with an association constant of (1.8 +/- 0.1)x10(4) M(-1). The ratio of the equilibrium association constants for HN3 and N3- binding provides a discrimination factor between the neutral and charged forms of the ligand. The discrimination factor is greater than 5800 for CcP but only nine for Mb. Protonation of the distal histidines in the two proteins influences binding of HN3. Protonation of His-64 in Mb enhances HN3 binding due to a gating mechanism while protonation of His-52 in CcP decreases the affinity for HN3 due to loss of base-assisted association of the ligand to the heme iron.
酵母细胞色素c过氧化物酶（CcP）和马高铁肌红蛋白（Mb）在25℃时以相似的亲和力结合叠氮酸（HN3）。形成CcP.HN3和Mb.HN3复合物的与pH无关的平衡缔合常数分别为(1.05±0.06)×10⁵和(1.6±0.8)×10⁵ M⁻¹。然而，形成这两种复合物的热力学参数有很大差异。形成CcP.HN3和Mb.HN3的ΔH0值分别为-16.4±0.7和-9.0±0.5 kcal/mol，ΔS0值分别为-32±2和-16±2 cal/deg mol。与HN3相关的质子在低pH时保留在两种蛋白质复合物中，但对于Mb.HN3和CcP.HN3复合物，其解离的表观pKA值分别为5.5±0.2和≥8.2。CcP和Mb对叠氮阴离子N3⁻的反应性有显著差异。CcP与N3⁻的结合非常弱（如果有结合的话），对于CcP.N3⁻复合物，只能确定其与pH无关的平衡缔合常数的上限为18±5 M⁻¹。Mb与N3⁻的缔合常数为(1.8±0.1)×10⁴ M⁻¹。HN3和N3⁻结合的平衡缔合常数之比提供了配体中性和带电形式之间的区分因子。对于CcP，区分因子大于5800，而对于Mb仅为9。两种蛋白质中远端组氨酸的质子化影响HN3的结合。Mb中His-64的质子化由于门控机制增强了HN3的结合，而CcP中His-52的质子化由于配体与血红素铁的碱辅助缔合丧失而降低了对HN3的亲和力。
