NMR investigations of the Rieske protein from Thermus thermophilus support a coupled proton and electron transfer mechanism.
NMR investigations of the Rieske protein from Thermus thermophilus support a coupled proton and electron transfer mechanism.
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DOI：
10.1021/ja1026387
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发表时间：
2010-06-16
影响因子：
15
通讯作者：
Markley, John L.
中科院分区：
文献类型：
--
作者：
Hsueh, Kuang-Lung;Westler, William M.;Markley, John L.
细胞色素bc复合物的Rieske蛋白质成分包含一个由两个半胱氨酸和两个组氨酸连接的[2Fe−2S]簇。我们在这里报告的pKa值的两个连接组氨酸（His 134和His 154）的Rieske蛋白的氧化和还原状态从嗜热栖热菌（TtRp）的每个咪唑环的NMR光谱测定。这些pKa值的知识是至关重要的兴趣，因为它们的相关性的分叉Q-循环中的电子和质子转移的机制。虽然我们之前已经观察到氧化TtRp（，，10672−10673）中两个配体组氨酸的15 N NMR信号的pH依赖性，但[2Fe−2S]簇的强顺磁性阻止了这些信号通过常规方法的分配。我们的方法是利用独特的组氨酸-亮氨酸（His 134-Leu 135）序列，并使用残基选择性标记来建立关键的序列特异性分配，然后将其扩展。对来自两个组氨酸簇配体的指定的13 C ′、13 C α和15 N ε2信号的pH依赖性分析导致氧化和还原TtRp的pKa值的明确指定。结果表明，His 134的pKa从氧化态的9.1变化到还原态的12.3，而His 154的pKa从氧化态的7.4变化到还原态的12.6。这确立了当Rieske蛋白位于细胞色素B位点时接近醌的His 154作为经历显著的氧化还原依赖性pKa位移的残基。氧化和还原TtRp的二级结构分析的基础上，我们广泛的化学位移分配排除了氧化和还原状态之间的大的构象变化。因此，TtRp可能通过被动扩散在细胞色素B和细胞色素c位点之间易位。我们的研究结果是最一致的一种机制，涉及耦合转移的电子和转移的质子之间的氢键的氢醌和His 154在细胞色素B网站。
The Rieske protein component of the cytochrome bc complex contains a [2Fe−2S] cluster ligated by two cysteines and two histidines. We report here the pKa values of each of the imidazole rings of the two ligating histidines (His134 and His154) in the oxidized and reduced states of the Rieske protein from Thermus thermophilus (TtRp) as determined by NMR spectroscopy. Knowledge of these pKa values is of critical interest because of their pertinence to the mechanism of electron and proton transfer in the bifurcated Q-cycle. Although we earlier had observed the pH dependence of a 15N NMR signal from each of the two ligand histidines in oxidized TtRp (, , 10672−10673), the strong paramagnetism of the [2Fe−2S] cluster prevented the assignment of these signals by conventional methods. Our approach here was to take advantage of the unique histidine−leucine (His134−Leu135) sequence and to use residue-selective labeling to establish a key sequence-specific assignment, which was then extended. Analysis of the pH dependence of assigned 13C′, 13Cα, and 15Nε2 signals from the two histidine cluster ligands led to unambiguous assignment of the pKa values of oxidized and reduced TtRp. The results showed that the pKa of His134 changes from 9.1 in oxidized to ∼12.3 in reduced TtRp, whereas the pKa of His154 changes from 7.4 in oxidized to ∼12.6 in reduced TtRp. This establishes His154, which is close to the quinone when the Rieske protein is in the cytochrome b site, as the residue experiencing the remarkable redox-dependent pKa shift. Secondary structural analysis of oxidized and reduced TtRp based upon our extensive chemical shift assignments rules out a large conformational change between the oxidized and reduced states. Therefore, TtRp likely translocates between the cytochrome b and cytochrome c sites by passive diffusion. Our results are most consistent with a mechanism involving the coupled transfer of an electron and transfer of the proton across the hydrogen bond between the hydroquinone and His154 at the cytochrome b site.
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