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首页> 外文期刊>Biochemistry >Effect of Hydrogen-Bond Networks in Controlling Reduction Potentials in Desulfovibrio vulgaris (Hildenborough) Cytochrome c_3 Probed by Site-Specific Mutagenesis
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Effect of Hydrogen-Bond Networks in Controlling Reduction Potentials in Desulfovibrio vulgaris (Hildenborough) Cytochrome c_3 Probed by Site-Specific Mutagenesis

机译：氢键网络对寻常脱硫弧菌（希尔登伯勒）细胞色素c_3还原电位控制的位点特异性诱变研究

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摘要

Cytochromes C3 isolated from Desulfovibrio spp. are periplasmic proteins that playa central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized and reduced structures of cytochrome C3 isolated from Desulfovibrio vulgaris (Hildenborough) show that the residue threonine 24, located in the vicinity of heme III, reorients between these two states [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, I., Turner, D. L., Santos, H., and Xavier, A. V. (1998) I. Mol. Riol. 281,719-739]. Threonine 24 was replaced with valine by site- directed mutagenesis to elucidate its effect on the redox properties of the protein. The NMR spectra of the mutated protein are very similar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the microscopic reduction potential of heme III (75 and 106 mV for the protonated forms of the fully reduced and oxidized states, respectively). The redox interactions involving this heme are also modified, while the remaining heme-heme interactions and the redox- Bohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is different from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by valine preventing the formation of a network of hydrogen bonds, which stabilizes the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbalances the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer and may affect the functionality of the protein.
机译：从Desulfovibrio spp分离的细胞色素C3。是周质蛋白，通过耦合来自氢化酶的电子和质子的传递，在能量转换中起着核心作用。从寻常脱硫弧菌（Hildenborough）分离出的细胞色素C3的氧化结构和还原结构之间的比较表明，位于血红素III附近的苏氨酸24残基在这两个状态之间重新定位[Messias，AC，Kastrau，DHW，Costa，HS， LeGall，I.，Turner，DL，Santos，H。和Xavier，AV（1998）I.Mol。瑞欧281,719-739]。通过定点诱变将苏氨酸24替换为缬氨酸，以阐明其对蛋白质氧化还原特性的影响。突变蛋白的NMR光谱与野生型非常相似，表明一般的折叠和血红素核心结构不受突变影响。但是，对突变的细胞色素的热力学分析表明，血红素III的微观还原电位有很大变化（对于完全还原和氧化态的质子化形式分别为75和106 mV）。涉及该血红素的氧化还原相互作用也被修饰，而其余的血红素-血红素相互作用和氧化还原-玻尔相互作用受到的影响较小。因此，突变的细胞色素中血红素的氧化顺序与野生型中的血红素的氧化顺序不同，并且它对电子的总体亲和力更高。这与用缬氨酸替代苏氨酸24防止形成氢键网络稳定了氧化态相一致。突变的蛋白质无法在中间氧化阶段1和3之间执行一致的两电子步骤，这可以在野生型蛋白质中发生。因此，替换单个残基不平衡调整为热力学控制逐步电子转移的方向性的全局协同网络，并且可能影响蛋白质的功能。

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