Effect of hydrogen-bond networks in controlling reduction potentials in Desulfovibrio vulgaris (Hildenborough) cytochrome C3 probed by site-specific mutagenesis.
Effect of hydrogen-bond networks in controlling reduction potentials in Desulfovibrio vulgaris (Hildenborough) cytochrome C3 probed by site-specific mutagenesis.
复制标题
通过定点诱变探测氢键网络在控制脱硫弧菌 (Hildenborough) 细胞色素 C3 还原电位中的作用。
作者：
C. Salgueiro;P. N. da Costa;D. Turner;A. C. Messias;W. V. van Dongen;L. M. Saraiva;A. V. Xavier
从脱硫弧菌属 (Desulfovibrio spp.) 中分离出细胞色素 C3。是周质蛋白，通过耦合来自氢化酶的电子和质子的转移，在能量转导中发挥核心作用。从 Desulfovibrio vulgaris (Hildenborough) 分离的细胞色素 C3 的氧化和还原结构之间的比较表明，位于血红素 III 附近的残基苏氨酸 24 在这两种状态之间重新定向 [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., 和泽维尔，A.V. (1998) J. Mol。生物。 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。突变蛋白的核磁共振谱与野生型非常相似，表明一般折叠和血红素核心结构不受突变影响。然而，突变细胞色素的热力学分析揭示了血红素 III 的微观还原电位的巨大变化（完全还原和氧化状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改，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，突变细胞色素中血红素的氧化顺序与野生型不同，并且对电子具有更高的总体亲和力。这与苏氨酸 24 被缬氨酸取代防止形成氢键网络一致，从而稳定了氧化态。突变蛋白无法在中间氧化阶段 1 和 3 之间执行协调的双电子步骤，而野生型蛋白可能会出现这种情况。因此，替换单个残基会导致全局协同网络失衡，从而在热力学上控制逐步电子转移的方向性，并可能影响蛋白质的功能。
Cytochromes C3 isolated from Desulfovibrio spp. are periplasmic proteins that play a 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, J., Turner, D. L., Santos, H., and Xavier, A. V. (1998) J. Mol. Biol. 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.