Min T, Ergenekan C E, Eidsness M K, Ichiye T, Kang C
School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660, USA.
Protein Sci. 2001 Mar;10(3):613-21. doi: 10.1110/gad.34501.
Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated. First, expansion of the [Fe-S] cluster and concomitant contraction of the NH...S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high-resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin.
生物电子转移是一个高效的过程，尽管氧化还原部分之间的距离通常相当大。因此，了解这些反应的速率和驱动力的物理基础具有重要意义。氧化还原状态变化时蛋白质发生的结构弛豫产生了重组能，这对所涉及蛋白质的速率和驱动力很重要。为了确定氧化还原蛋白中的结构弛豫，我们开发了一些方法，在结晶过程中将氧化还原蛋白保持在其最终氧化态，同时维持其初始氧化态结晶时相同的pH值和盐浓度条件。基于来自巴氏梭菌（Cp）的氧化型和还原型红素氧还蛋白（Rd）的1.5埃分辨率晶体结构和分子动力学模拟，还原时的结构重排表明了促进红素氧还蛋白电子转移反应的具体机制。首先，[Fe-S]簇的扩张以及NH...S氢键的相应收缩导致额外负电荷的静电稳定增强。其次，由非极性侧链亮氨酸41的构象变化引起的门控机制允许水分子短暂渗透，这极大地增加了氧化还原位点环境的极性，也提供了质子源。我们从还原溶液中制备Cp Rd晶体的方法导致了在激烈火球菌还原型Rd晶体结构中未观察到的水分子分布。这种相关性在氧化还原蛋白中普遍程度如何，必须在未来的工作中确定。我们高分辨率晶体结构和分子动力学模拟的结合提供了Cp红素氧还蛋白还原时发生的结构重排的分子图景。
