Redox and conformational equilibria and dynamics of cytochrome c at high electric fields.
Redox and conformational equilibria and dynamics of cytochrome c at high electric fields.
复制标题
DOI：
10.1002/cphc.200200618
复制
发表时间：
2003-07
期刊：
影响因子：
--
通讯作者：
H. Wackerbarth;P. Hildebrandt
中科院分区：
文献类型：
--
作者：
H. Wackerbarth;P. Hildebrandt
用稳态和时间分辨表面增强共振拉曼光谱研究了吸附在银电极/电解液界面双电层中的细胞色素c(Cyt-c)，分析了强电场对蛋白质结构、反应平衡和动力学的影响。在+0.1~-0.55 V(相对于饱和甘汞电极)的电势范围内，吸附的Cyt-c在自然态B1和构象态B2之间形成了一个依赖于电位的可逆平衡。双组氨酸配位的六配位低自旋和五配位高自旋亚态的氧化还原电位分别为-0.425和-0.385 V，而另外六配位的水组氨酸配位的高自旋亚态则是氧化还原失活的。构象状态B1的氧化还原电势与溶液中的相同，与吸附的B1和天然Cyt-c的结构一致。对于所有三个氧化还原活性物种，形式非均相电子转移速率常数都很小，且具有相同的数量级(3-13 S-1)，这意味着速率限制步骤在很大程度上与氧化还原位结构无关。这些发现，以及各种构象(亚)态之间缓慢且依赖于电位的转变，可以用电场诱导的与蛋白质结构重组相关的质子转移步骤的激活能增加来解释。通过将电极电位移动到+0.1V以上来进一步增加电场强度会导致不可逆的结构变化，这归因于多肽链的展开。
Cytochrome c (Cyt-c) adsorbed in the electrical double layer of the Ag electrode/electrolyte interface has been studied by stationary and time-resolved surface-enhanced resonance Raman spectroscopy to analyse the effect of strong electric fields on structure and reaction equilibria and dynamics of the protein. In the potential range between +0.1 and -0.55 V (versus saturated calomel electrode), the adsorbed Cyt-c forms a potential-dependent reversible equilibrium between the native state B1 and a conformational state B2. The redox potentials of the bis-histidine-coordinated six-coordinated low-spin and five-coordinated high-spin substates of B2 were determined to be -0.425 and -0.385 V, respectively, whereas the additional six-coordinated aquo-histidine-coordinated high-spin substate was found to be redox-inactive. The redox potential for the conformational state B1 was found to be the same as in solution in agreement with the structural identity of the adsorbed B1 and the native Cyt-c. For all three redox-active species, the formal heterogeneous electron transfer rate constants are small and of the same order of magnitude (3-13 s-1), which implies that the rate-limiting step is largely independent of the redox-site structure. These findings, as well as the slow and potential-dependent transitions between the various conformational (sub-)states, can be rationalized in terms of an electric field-induced increase of the activation energy for proton-transfer steps linked to protein structural reorganisation. Further increasing the electric field strength by shifting the electrode potential above +0.1 V leads to irreversible structural changes that are attributed to an unfolding of the polypeptide chain.