A New Class of Integral Membrane Oxidoreductases

一类新型整体膜氧化还原酶

• 批准号: 0646621
• 负责人: Robert Blankenship
• 金额: $ 60万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0646621&HistoricalAwards=false
• 关键词: New; Class; Integral; Membrane; Oxidoreductases

A new class of integral membrane oxidoreductase complex was recently discovered in bacteria (Yanyushin et al. Biochemistry, 44, 10037-10045, 2005). The complex was purified from the filamentous green anoxygenic phototroph Chloroflexus aurantiacus. The complex consists of seven subunits, the genes for which were identified using trypsin disgestion fingerprinting, mass spectroscopy and the partially completed genome sequence. Three of the subunits appear to be integral membrane proteins based on hydropathy plots. Two of the subunits contain c-type heme cofactors, based on both heme staining and sequence data. Another subunit has sequence homology at the N-terminal end to a molybdopterin enzyme and the C-terminal end to an FeS protein, although the complex does not contain Mo or pterin cofactors, and the primary sequence is missing key elements of their binding sites. The complex has been named the MFIcc complex. Similar although distinct complexes were isolated from phototrophically and aerobically grown cells, and appear to be coded for by distinct genes for at least most of the subunits. The genes that code for the protein subunits of the complexes are arranged in a cluster on the genome and form a putative operon. What appear to be homologous operons for similar complexes were identified in genomes of a number of nonphotosynthetic bacteria. The presence of genes for this complex are anticorrelated with the presence of genes for the cytochrome bc1 complex, suggesting that the new complex may functionally replace the cytochrome bc1 complex. Chloroflexus aurantiacus almost certainly does not contain a bc1 complex based on both biochemical studies and the lack of genes in its genome that code for the protein subunits. This project involves complete biochemical and energetic characterization of this complex. This includes determination of overall oligomeric state and numbers of each subunit by the use of gel filtration and blue-native and SDS gel electrophoresis, coupled with mass spectroscopy and protein cross-linking studies. Additional information about the redox centers and their midpoint potentials will be obtained using redox potentiometry coupled with UV-Vis and EPR spectroscopies. Quantitative determination of the number and types of cofactors will be carried out using a variety of standard techniques. Broader ImpactsThe information obtained on these complexes will provide new insights into novel bacterial bioenergetic mechanisms and the origin and evolution of energy conserving systems. This project will train a graduate student and a postdoctoral fellow in the integrated techniques of biochemistry, molecular biology, biophysical analysis and bioinformatics that are essential parts of modern research in bioenergetic systems. Undergraduate students will also be involved in all aspects of the research.

最近在细菌中发现了一种新的一类综合膜氧化还原酶复合物（Yanyushin等人生物化学，44，10037-10045，2005）。该复合物是从丝状绿色无氧光链球冠状动脉上纯化的。该复合物由七个亚基组成，这些亚基是使用胰蛋白酶旋转指纹，质谱和部分完成的基因组序列鉴定的基因。三个亚基似乎是基于水性质图的整体膜蛋白。基于血红素染色和序列数据，两个亚基包含C型血红素辅因子。另一个亚基在N末端具有序列的序列同源性，与FES蛋白的C末端与C末端端具有序列同源性，尽管该复合物不含MO或PTERIN辅助因子，并且主要序列缺少其结合位点的关键元素。该综合体已命名为MFICC复合体。相似的虽然从光养和有氧生长的细胞中分离出不同的复合物，并且似乎至少在大多数亚基中被不同的基因编码。为复合物的蛋白质亚基编码的基因在基因组上的簇中排列并形成推定的操纵子。在多种非肠道细菌的基因组中鉴定出似乎是类似复合物的同源操纵子。该复合物的基因的存在与细胞色素BC1复合物的基因的存在是反相关的，这表明新复合物可以在功能上取代细胞色素BC1复合物。基于生物化学研究和其基因组中缺乏基因的基因组，氯曲叶曲lixususususus几乎肯定不包含BC1复合物，以编码蛋白质亚基。该项目涉及该复合物的完整生化和能量表征。这包括通过使用凝胶过滤和蓝色和SDS凝胶电泳来确定总体寡聚状态和每个亚基的数量，并与质谱和蛋白质交联研究结合使用。有关氧化还原中心及其中点电势的其他信息将使用与UV-VIS和EPR光谱镜结合的氧化还原电位测量法获得。将使用各种标准技术对辅助因子的数量和类型进行定量测定。更广泛的影响这些复合物获得的信息将为新的细菌生物能机制以及能源养生系统的起源和演变提供新的见解。该项目将培训研究生和博士后研究员的生物化学，分子生物学，生物物理分析和生物信息学的综合技术，这些技术是生物能系统中现代研究的重要组成部分。本科生还将参与研究的各个方面。