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类新的整合膜氧化还原酶复合物（Yanyushin等人，Biochemistry，44，10037-10045，2005）。该复合物是从丝状绿色厌氧光养菌Chloroflexus aurantiacus中纯化得到的。该复合物由七个亚基组成，其基因使用胰蛋白酶消化指纹图谱、质谱和部分完成的基因组序列进行鉴定。三个亚基似乎是基于亲水性图的整合膜蛋白。根据血红素染色和序列数据，其中两个亚基含有c型血红素辅因子。另一个亚基在N-末端与蝶呤酶具有序列同源性，在C-末端与FeS蛋白具有序列同源性，尽管复合物不含Mo或蝶呤辅因子，并且一级序列缺少其结合位点的关键元件。该复合物被命名为MFIcc复合物。类似的，但不同的复合物分离自光养和需氧生长的细胞，似乎是由不同的基因编码的至少大部分的亚基。编码复合物蛋白质亚基的基因在基因组上排列成簇，形成一个假定的操纵子。在一些非光合细菌的基因组中发现了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bc 1复合物的基因的存在是相关的，这表明新的复合物可能在功能上取代细胞色素bc 1复合物。根据生化研究和其基因组中编码蛋白质亚基的基因的缺失，橙色绿弯藻几乎可以肯定不含有bc 1复合体。该项目涉及对该复合体的完整生物化学和能量表征。这包括通过使用凝胶过滤和蓝色-天然和SDS凝胶电泳，结合质谱和蛋白质交联研究，确定每个亚基的总体寡聚状态和数量。关于氧化还原中心及其中点电位的其他信息将使用氧化还原电位法结合UV-Vis和EPR光谱法获得。辅因子的数量和类型的定量测定将使用各种标准技术进行。更广泛的影响在这些复合物上获得的信息将为新的细菌生物能量机制以及节能系统的起源和进化提供新的见解。该项目将培养一名研究生和一名博士后研究员，掌握生物化学、分子生物学、生物物理分析和生物信息学的综合技术，这些技术是生物能量系统现代研究的重要组成部分。本科生也将参与研究的各个方面。