Similarities in structure between holocytochrome b5 and apocytochrome b5: NMR studies of the histidine residues.
Similarities in structure between holocytochrome b5 and apocytochrome b5: NMR studies of the histidine residues.
复制标题
全细胞色素 b5 和脱辅基细胞色素 b5 之间结构的相似性：组氨酸残基的 NMR 研究。
DOI：
10.1021/bi00098a012
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Lecomte,JT
中科院分区：
文献类型：
--
作者：
Moore,CD;al-Misky,ON;Lecomte,JT
化学系，宾夕法尼亚州立大学，大学公园，宾夕法尼亚16802接收1991年4月1日;修订的Mandarin pt接收1991年6月13日摘要：通过使用一维和二维质子核磁共振光谱研究了脱辅基细胞色素b5的六个组氨酸残基的性质，以探测血红素去除后的结构变化。光谱分配是通过分析质子NOE连接性，将它们与在全蛋白中观察到的进行比较，并检查后者物种的X射线结构来实现的。研究了每个组氨酸残基的pKa值、与松弛剂次氮基三乙酸铜的相互作用以及对溴乙酸的反应性。在脱辅基蛋白的主要构象中，没有与全蛋白中的铁原子配位的四个组氨酸（His-15、-26、-27和-80）显示出与全蛋白中相同的特征性质。其中三个参与与结构其余部分的特定相互作用：His-15和His-80参与氢键，His-27受附近C-末端片段的影响。His-26是暴露于溶剂中最多的。位于血红素结合位点的His-63和His-39具有不同的pKa值;它们受铜试剂的影响不同，并且对溴乙酸表现出相当的反应性，尽管比His-26温和。结果表明，血红素结合残基是明确区分其物理化学性质和天然的全蛋白结构的几个元素在脱辅基蛋白的地方。有人建议，血红素的结构影响是本地化的，氨基和羧基末端的片段形成一个结构单元，提供稳定的脱辅基蛋白和支持波动，部分折叠的结合位点。细胞色素B y的含血红素的水溶性片段（细胞色素B y是由α-螺旋和β-折叠组成的小球状蛋白质（马修斯等人，1971年）。它的血红素辅基埋在蛋白质基质中，已知在全蛋白结构的定义和稳定中起重要作用（亨特利和Strittmatter，1972）。为了探索这一作用，我们目前正在通过核磁共振光谱表征载脂蛋白的理化性质。我们最近报道的结果表明，在脱辅基蛋白中存在一个稳定的侧链簇，它与全蛋白的第二个疏水核心重叠（摩尔和勒孔特，1990）。这个结构化区域包含唯一的色氨酸残基Trp-22和蛋白质的六个组氨酸残基中的两个His-15和His-80。在全蛋白中，His-15和His-80被发现在β-折叠的相对侧，与骨架原子形成氢键，并且被认为分别稳定螺旋I和VI（马修斯等人，1979年）。其他四个组氨酸残基位于26、27、39和63位。His-26和His-27位于连接链3和4的一个转弯处; His-39和His-63是铁原子的两个轴向配体，并终止形成血红素腔的四个螺旋中的两个。因此，六个组氨酸残基分散在整个蛋白质中，并提供了良好的结构特征标记。特别地，部分地由美国国立卫生研究院的生物医学研究支持资助计划授予的BRSGGrant S 07 RR 07082 -22和部分地由美国国立卫生研究院的Grant DK 43101支持的两种血红素f-
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 Received April 1, 1991; Revised Manuscript Received June 13, 1991 abstract: The properties of the six histidine residues of apocytochrome b5 have been investigated by using one-and two-dimensional proton NMR spectroscopy in orderto probe the structure remainingafter heme removal. Spectral assignments were arrived at by analyzing proton NOE connectivities, comparing them to those observed in the holoprotein, and inspecting the X-ray structure of the latter species. Each histidine residue was studied for its pKa value, interactionwith the relaxation agent copper nitrilotriacetic acid, and reactivity toward bromoacetic acid. Thefour histidines which are not coordinated to the iron atom in the holoprotein (His-15,-26,-27, and-80) display in the major conformer of the apoprotein the same characteristic properties as in the holoprotein. Three of them are involved in specific interactions with the rest of the structure: His-15 and His-80 participate in hydrogen bonds, and His-27 is influenced by the nearby C-terminal segment. His-26 is the most exposed to the solvent. His-63 and His-39, which are located in the heme binding site, have distinct pKa values; they are affected differently by the copper agent and exhibit comparable reactivity toward bromoaceticacid, albeit milder than that of His-26. Theresults show that the heme binding residues are clearly distinguishable by their physicochemical propertiesand that several elements of native holoprotein structure are in place in the apoprotein. It is proposed that the structural influence of the heme is localized and that the amino-and carboxy-terminal segments form a structural unit providing stability to the apoprotein and supporting a fluctuating, partially folded binding site. e heme-containing, water-soluble fragment of cytochrome b¡(cyt b} y is a small globular protein made of both a-helices and/3-sheet (Mathews et al., 1971). Its heme prosthetic group is buried in the protein matrix and is known to play an essential role in the definition and stabilization of the structure of the holoprotein (Huntley & Strittmatter, 1972). In order to probe this role, we are currently characterizing physicochemical properties of the apoprotein by nuclear magnetic resonance spectroscopy. We recently reported results demonstrating the presence in the apoprotein of a stable cluster of side chains which overlaps with the second hydrophobic core of the holoprotein (Moore & Lecomte, 1990). This structured region contains the only tryptophan residue, Trp-22, and two of the protein’s six histidine residues, His-15 and His-80. In the holoprotein, His-15 and His-80 are found on opposite sides of the/3-sheet, form hydrogenbonds with backbone atoms, and are thought to stabilize helices I and VI, respectively (Mathews et al., 1979). The otherfour histidine residues are located at positions 26, 27, 39, and 63. His-26 and-27 are situated in a turn connecting strands 3 and 4; His-39 and His-63 are the two axial ligands of the iron atom and terminate two of the four helices forming theheme cavity. Thus, the six histidine residues are scattered throughout the protein and providegood markers of structural features. In particular, the two heme f Supported in part by BRSGGrant S07 RR07082-22 awarded by the Biomedical Research Support Grant Program of the National Institutes of Health and in part by Grant DK 43101 from the National Institutes of Health-