Department of Chemistry , Dartmouth College , Hanover , New Hampshire 03755 , United States.
Department of Chemistry and Biochemistry , Berry College , Mount Berry , Georgia 30149 , United States.
Inorg Chem. 2019 Oct 21;58(20):14085-14106. doi: 10.1021/acs.inorgchem.9b02111. Epub 2019 Oct 7.
Ligand-switch reactions at the heme iron are common in biological systems, but their mechanisms and the features of the polypeptide fold that support dual ligation are not well understood. In cytochrome (cyt ), two low-stability loops (Ω-loop C and Ω-loop D) are connected by the heme propionate HP6. At alkaline pH, the native Met80 ligand from Ω-loop D switches to a Lys residue from the same loop. Deprotonation of an as yet unknown group triggers the alkaline transition. We have created the two cyt variants T49V/K79G and T78V/K79G with altered connections of these two loops to HP6. Electronic absorption, NMR, and EPR studies demonstrate that at pH 7.4 ferric forms of these variants are Lys-ligated, whereas ferrous forms maintain the native Met80 ligation. Measurements of protein stability, cyclic voltammetry, pH-jump and gated electron-transfer kinetics have revealed that these Thr to Val substitutions greatly affect the alkaline transition in both ferric and ferrous proteins. The substitutions modify the stability of the Met-ligated species and reduction potentials of the heme iron. The kinetics of ligand-switch processes are also altered, and analyses of these effects implicate redox-dependent differences in metal-ligand interactions and the role of the protein dynamics, including cross-talk between the two Ω-loops. With the two destabilized variants, it is possible to map energy levels for the Met- and Lys-ligated species in both ferric and ferrous proteins and assess the role of the protein scaffold in redox-dependent preferences for these two ligands. The estimated shift in the heme iron reduction potential upon deprotonation of the "trigger" group is consistent with those associated with deprotonation of an HP, suggesting that HP6, on its own or as a part of a hydrogen-bonded cluster, is a likely "trigger" for the Met to Lys ligand switch.
配体在血红素铁的开关反应在生物系统中很常见，但它们的机制和支持双重配体的多肽折叠特征还不太清楚。在细胞色素 c（cyt c）中，两个低稳定性环（Ω-环 C 和 Ω-环 D）通过血红素丙酸酯 HP6 连接。在碱性 pH 值下，来自 Ω-环 D 的天然 Met80 配体切换到来自同一环的 Lys 残基。一个未知的基团的去质子化触发了碱性转变。我们已经创建了两个 cyt c 变体 T49V/K79G 和 T78V/K79G，它们的这两个环与 HP6 的连接发生了改变。电子吸收、NMR 和 EPR 研究表明，在 pH 值为 7.4 时，这些变体的 ferric 形式是 Lys 配位的，而 ferrous 形式保持天然的 Met80 配位。蛋白质稳定性测量、循环伏安法、pH 跳跃和门控电子转移动力学测量表明，这些 Thr 到 Val 的取代极大地影响了 ferric 和 ferrous 蛋白中的碱性转变。这些取代改变了 Met 配位物种的稳定性和血红素铁的还原电位。配体开关过程的动力学也发生了改变，对这些效应的分析表明金属-配体相互作用的氧化还原依赖性差异以及蛋白质动力学的作用，包括两个 Ω-环之间的串扰。使用两个不稳定的变体，可以绘制 ferric 和 ferrous 蛋白中 Met 和 Lys 配位物种的能级，并评估蛋白质支架在氧化还原依赖性对这两种配体的偏好中的作用。“触发”基团去质子化时血红素铁还原电位的估计位移与与 HP 去质子化相关的那些位移一致，这表明 HP6 本身或作为氢键簇的一部分，可能是 Met 到 Lys 配体开关的“触发”。
