Identification of the predominant non-native histidine ligand in unfolded cytochrome c
Identification of the predominant non-native histidine ligand in unfolded cytochrome c
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DOI：
10.1021/bi971697c
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发表时间：
1997-10-14
期刊：
影响因子：
2.9
通讯作者：
Roder, H
中科院分区：
文献类型：
--
作者：
Colon, W;Wakem, LP;Roder, H
血红素及其两个轴向配体His 18和Met 80在细胞色素c的折叠/解折叠机制中起着核心作用。由于共价血红素连接，His 18在典型的变性条件下保持结合，而更不稳定的Met 80配体被替代的组氨酸配体取代。为了区分马细胞色素c中的两种可能的非天然组氨酸配体，使用酵母表达系统制备具有His 26至Gln或His 33至Asn取代的变体。在盐酸胍变性状态下的非天然组氨酸配体的质子化导致Soret血红素吸收带的明显蓝移（低自旋到高自旋的转变）。而His 26的取代对该转变的表观pK（a）没有影响（5.7 +/- 0.05），H33 N变体表现出显著更高的pK（a）（6.1 +/- 0.05），表明His 33是变性细胞色素c中占优势的第六个血红素配体，His 26在位置33处不存在组氨酸的情况下，含氮基团（或另一含氮基团）充当配体。pH诱导的配体解离的动力学显示了两个阶段，这两个组氨酸配体的基础上，其不同的温度依赖性被分配给每个。尽管它们几乎相同的平衡展开转换，两个组氨酸突变体显示其折叠动力学的差异。虽然H26 Q cyt c的动力学行为与野生型非常相似，但H33 N突变导致动力学相的丧失，速率在2-10 s（-1）范围内，先前已将其归因于捕获的非天然组氨酸的限速解离，因此将其鉴定为His 33。
The heme and its two axial ligands, His18 and Met80, play a central role in the folding/ unfolding mechanism of cytochrome c. Because of the covalent heme attachment, His18 remains bound under typical denaturing conditions, while the more labile Met80 ligand is replaced by an alternate histidine ligand. To distinguish between the two possible non-native histidine ligands in horse cytochrome c, variants with a His26 to Gin or His33 to Asn substitution were prepared using a yeast expression system. Protonation of the non-native histidine ligand in the GuHCl-denatured state results in a pronounced blue shift of the Soret heme absorbance band (low-spin to high-spin transition). While substitution of His26 has no effect on the apparent pK(a) of this transition (5.7 +/- 0.05), the H33N variant exhibits a substantially higher pK(a) (6.1 +/- 0.05), indicating that His33 is the dominant sixth heme ligand in denatured cytochrome c and that His26 (or another nitrogenous group) acts as a ligand in the absence of a histidine at position 33. The kinetics of the pH induced ligand dissociation shows two phases which were assigned to each of the two histidine ligands on the basis of their distinct temperature dependence. Despite their nearly identical equilibrium unfolding transitions, the two histidine mutants show differences in their folding kinetics. While the kinetic behavior of H26Q cyt c is very similar to that of the wild-type, the H33N mutation leads to loss of a kinetic phase with a rate in the 2-10 s(-1) range that has previously been attributed to the rate-limiting dissociation of a trapped non-native histidine, which is thus identified as His33.