15N and 1H NMR studies of Rhodospirillum rubrum cytochrome c2.
15N and 1H NMR studies of Rhodospirillum rubrum cytochrome c2.
复制标题
红色红螺菌细胞色素 c2 的 15N 和 1H NMR 研究。
DOI：
10.1021/bi00406a022
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Smith,GM
中科院分区：
文献类型：
--
作者：
Yu,LP;Smith,GM
加州大学食品科学与技术系，戴维斯，加利福尼亚州 95616 1987 年 9 月 17 日收稿；修订稿收到 1987 年 11 月 17 日 摘要：从在 15NH4Cl 上生长的红色红螺菌中纯化出富集 15N 的细胞色素 c2，并通过 15N 和 NMR 光谱研究了细胞色素的抗磁性铁 (II) 形式。指定了四个吡咯氮、配体组氨酸氮、高度保守的色氨酸吲哚氮和一些脯氨酸氮的 15N 共振。由于质子互变异构产生的严重展宽，仅在低 pH 下观察到单个非配体组氨酸的共振。还指定了与配体组氨酸、色氨酸和一些酰胺基团的氮键合的可交换质子的共振。氮结合质子的交换率差异很大：大多数半衰期不到几分钟，Trp-62 的吲哚 NH 交换半衰期为数周，配体组氨酸 NH 质子交换半衰期为数月。后一个观察结果表明溶剂被极端排除在配体组氨酸周围的区域之外，并为暗示该区域的疏水性程度作为调节中点电位的重要因素的理论提供了依据。铁细胞色素 c2 的 15N 和 NMR 光谱对 pH 的依赖性表明，在 pH 9.5 以下，Trp-62 和配体 His 侧链都不会去质子化到任何明显的程度。在整个 pH 滴定过程中，His-18 NH 可能与 Pro-19 羧基保持氢键键合。由于在铁细胞色素的光谱中未观察到 His-18 的去质子化或非氢键形式，因此此类形式参与产生具有不同 g 张量值的异质群体似乎不太可能。 pK 为 6.8 时发生的单次电离会导致吡咯氮和血红素附近的几个基团的共振发生变化。这种电离归因于 His-42 或 His-42-血红素丙酸氢键对的质子化/去质子化，似乎影响血红素附近的广泛基团，可能是通过改变覆盖该蛋白质区域的环的堆积。 e 细胞色素 c2 是在紫色非硫细菌中发现的一类电子传递蛋白。它们在光合电子传输中的功能的重要性以及它们与线粒体细胞色素c的结构同源性使它们成为近年来大量研究的主题。许多结构和功能参数已被测量。这项研究得到了 NIH Grant GM-34194 的支持。* 通讯应寄给本作者。当然，同样许多尝试将结构与功能联系起来。对细胞色素明显重要的一个特性是血红素铁的中点氧化还原（氧化还原）电位。细胞色素的血红素基团的特征由蛋白质提供的配体和折叠肽链产生的环境决定。已经提出了几种关于细胞色素中点电位的控制和多样性的理论。
Department of Food Science and Technology, University of California, Davis, California 95616 Received September 17, 1987; Revised Manuscript Received November 17, 1987 abstract: 15N-Enriched cytochrome c2 was purified from Rhodospirillum rubrum that had been grown on 15NH4C1, andthe diamagnetic iron (II) form of the cytochrome was studied by 15N and NMR spectroscopy. 15N resonances of the four pyrrolenitrogens, the ligand histidine nitrogens, the highly conserved tryptophan indole nitrogen, and some proline nitrogens are assigned. The resonances of the single nonligand histidine are observed only at low pH because of severe broadening produced by proton tautomerization. The resonances of exchangeable protons bonded to the nitrogens of the ligand histidine, the tryptophan, and some amide groups are also assigned. The exchange rates of the nitrogen-bound protons vary greatly: most have half-lives of less than minutes, the indolic NH of Trp-62 exchanges with a half-time of weeks, and the ligand histidine NH proton exchanges with a half-time of months. The latter observation is indicative of extreme exclusion of solvent from the area surrounding the ligand histidine and lends credence to theories implicating the degree of hydrophobicity in this region as an important factor in adjusting the midpoint potential. Thedependence of the 15N and NMR spectra of ferrocytochrome c2 on pH indicates neither the Trp-62 nor the ligand His side chains become deprotonated to any appreciable extent below pH 9.5. The His-18 NH remains hydrogen bonded, presumablyto the Pro-19 carboxyl group, throughout the pH titrations. Because neither deprotonated nor non-hydrogen-bonded forms of His-18 are observed in spectra of the ferrocytochrome, the participation of such formsin producing a heterogeneous population having different g tensor values seems unlikely. A single ionization, occurring with a pK of 6.8, causes the resonances of the pyrrole nitrogens and several groups near the heme to shift. This ionization, attributed to the protonation/deprotonation of His-42 or a His-42-heme propionate hydrogen-bonded pair, appears to affect a wide range of groups near the heme, perhaps by altering the packing of the loops which cover that region of the protein. e cytochromes c2 are a class of electron-transport proteins found in the purple non-sulfur bacteria. The importance of their function in photosyntheticelectron transport and their structural homology to the mitochondrial cytochrome c have made them the subject of a great deal of study in recent years. Many structural and functional parameters have been mea-tThis research was supported by NIH Grant GM-34194.* Correspondence should be addressed to this author. sured, and equally many attempts have been made to relate structure to function.A property of obvious importance to the cytochromes is the midpoint oxidation-rt duction (redox) potential of theheme iron. The character of the heme groups of the cytochromes is determined both by the ligands provided by the protein and by the environment created by the folded peptide chains. Several theories concerning the control and diversity of the midpoint potentials of the cytochromes have been proposed.