Rivas Laura, Soares Cláudio M, Baptista António M, Simaan Jalila, Di Paolo Roberto E, Murgida Daniel H, Hildebrandt Peter
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Biophys J. 2005 Jun;88(6):4188-99. doi: 10.1529/biophysj.104.057232. Epub 2005 Mar 11.
The tetraheme protein cytochrome c(3) (Cyt-c(3)) from Desulfovibrio gigas, immobilized on a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid, is studied by theoretical and spectroscopic methods. Molecular dynamics simulations indicate that the protein docks to the negatively charged SAM via its lysine-rich domain around the exposed heme IV. Complex formation is associated with only little protein structural perturbations. This finding is in line with the resonance Raman and surface-enhanced resonance Raman (SERR) spectroscopic results that indicate essentially the same heme pocket structures for the protein in solution and adsorbed on SAM-coated Ag electrodes. Electron- and proton-binding equilibrium calculations reveal substantial negative shifts of the redox potentials compared to the protein in solution. The magnitude of these shifts decreases in the order heme IV (-161 mV) > heme III (-73 mV) > heme II (-57 mV) > heme I (-26 mV), resulting in a change of the order of reduction. These shifts originate from the distance-dependent electrostatic interactions between the SAM headgroups and the individual hemes, leading to a stabilization of the oxidized forms. The results of the potential-dependent SERR spectroscopic analyses are consistent with the theoretical predictions and afford redox potential shifts of -160 mV (heme IV), -90 mV (heme III), -70 mV (heme II), and +20 mV (heme I) relative to the experimental redox potentials for Cyt-c(3) in solution. SERR spectroscopic experiments reveal electric-field-induced changes of the redox potentials also for the structurally very similar Cyt-c(3) from Desulfovibrio vulgaris, although the shifts are somewhat smaller compared to Cyt-c(3) from D. gigas. This study suggests that electric-field-induced redox potential shifts may also occur upon binding to biomembranes or partner proteins and thus may affect biological electron transfer processes.
通过理论和光谱方法研究了固定在11-巯基十一烷酸自组装单分子层（SAM）上的来自巨大脱硫弧菌的四血红素蛋白细胞色素c(3)（Cyt-c(3)）。分子动力学模拟表明，该蛋白通过其暴露的血红素IV周围富含赖氨酸的结构域与带负电荷的SAM对接。复合物的形成仅伴随着很少的蛋白质结构扰动。这一发现与共振拉曼光谱和表面增强共振拉曼（SERR）光谱结果一致，这些结果表明溶液中的蛋白质和吸附在SAM包覆的银电极上的蛋白质具有基本相同的血红素口袋结构。电子和质子结合平衡计算表明，与溶液中的蛋白质相比，氧化还原电位有显著的负向移动。这些移动的幅度按血红素IV（-161 mV）>血红素III（-73 mV）>血红素II（-57 mV）>血红素I（-26 mV）的顺序降低，导致还原顺序发生变化。这些移动源于SAM头基与各个血红素之间的距离依赖性静电相互作用，导致氧化形式的稳定。电位依赖的SERR光谱分析结果与理论预测一致，相对于溶液中Cyt-c(3)的实验氧化还原电位，提供了-160 mV（血红素IV）、-90 mV（血红素III）、-70 mV（血红素II）和+20 mV（血红素I）的氧化还原电位移动。SERR光谱实验还揭示了电场诱导的来自普通脱硫弧菌的结构非常相似的Cyt-c(3)的氧化还原电位变化，尽管与来自巨大脱硫弧菌的Cyt-c(3)相比，移动幅度稍小。这项研究表明，电场诱导的氧化还原电位移动在与生物膜或伴侣蛋白结合时也可能发生，因此可能影响生物电子传递过程。
