BIOLOGICAL ELECTRON TRANSFER

在我们总体目标的背景下，了解 C型生物电子转移的结构和动力学研究 将进行细胞色素和其他氧化还原蛋白的研究。我们有 成功地开发了适用于定点定向的遗传系统 荚膜红杆菌细胞色素c2的诱变及其特性 一些变种人。到目前为止的研究主要集中在 电子转移动力学、氧化还原电位、蛋白质的突变 稳定性、蛋白质动力学和蛋白质结构。此外, 在表征结构特征方面取得了实质性进展 和氧化还原性质的高势铁硫蛋白和 细胞色素c‘。根据我们到目前为止的发现，我们建议 全面、完整的电子机制研究计划 初步利用细胞色素c2突变体进行遗传转化 与野生型和突变型光合作用反应中心的相互作用， 细胞色素c过氧化物酶和细胞色素c1。尽管主要关注点是 将对细胞色素c2，我们将进行并行，但不太广泛 与其他氧化还原蛋白家族如HIPIP，细胞色素c‘， 植物铁氧还蛋白和多血红素细胞色素。 我们正在将定点突变的使用与一系列 用分子术语定义电子转移的方法 细胞色素c2和电子供体和受体。因此，对这一问题的研究 电子转移动力学将与结构研究相结合(核磁共振 和x射线结晶学)，蛋白质稳定性的研究，氧化还原电位 和蛋白质动力学(核磁共振)。预计拟议的研究将 将使我们能够解决静电、立体学、距离的作用 氧化还原中心之间，相互作用的氧化还原中心的相对取向， 电子转移的中间媒介，以及蛋白质 生物电子转移中的动力学。拟议的研究还将 解决与控制生物氧化还原电位有关的问题， 特定氨基酸对结构域和蛋白质的重要性 稳定性，以及控制生物特异性和 调节蛋白质-蛋白质相互作用的识别。

In the context of our overall objective of understanding the mechanisms of biological electron transfer, both structural and kinetic studies on c-type cytochromes and other redox proteins will be carried out. We have successfully developed the appropriate genetic system for the site-directed mutagenesis of Rhodobacter capsulatus cytochrome c2 and have characterized a number of mutants. Studies to this point have focused on the effect of mutations on electron transfer kinetics, redox potential, protein stability, protein dynamics and protein structure. In addition, substantial progress has been made on characterization of the structural and redox of properties of the high potential iron sulfur proteins and the cytochromes c'. Based on our findings to date, we are proposing a comprehensive and integrated plan to investigate the mechanism of electron transfer primarily utilizing mutants of cytochrome c2 and by studying their interaction with both wild-type and mutant photosynthetic reaction centers, cytochrome c peroxidase and cytochrome c1. Although the principal focus will be on cytochrome c2, we will carry out parallel but less extensive efforts with other families of redox proteins such as HIPIP, cytochrome c', plant ferredoxin, and multi-heme cytochromes. We are combining the use of site-directed mutagenesis with a range of approaches in order to define in molecular terms electron transfer between cytochrome c2 and electron donors and acceptors. Thus, studies on the kinetics of electron transfer will be combined with structural studies (NMR and x-ray crystallography), studies on protein stability, redox potentials and protein dynamics (NMR). It is anticipated that the proposed studies will permit us to address the role of electrostatics, sterics, distance between redox centers, relative orientation of interacting redox centers, intervening media through which electrons are transferred, and protein dynamics in biological electron transfer. The proposed studies will also address issues related to the control of biological redox potentials, the importance of specific amino acids to structural domains, to protein stability, and the factors controlling biological specificity and recognition that mediate protein-protein interactions.