Reverse electron transfer involving a butyrate-induced membrane complex during syntrophic growth of Syntrophomonas wolfei

共生生长在厌氧环境中有机物的循环中起着至关重要的作用。合成代谢的一个显著特征是，由于临界氧化还原反应的不利热力学，需要反向电子转移。最近对专性厌氧模式生物沃尔夫合单胞菌和嗜酸营养盐合单胞菌的基因组学和蛋白质组学研究提供了证据，证明在丁酸盐的共生生长过程中诱导了etf连接的氢化酶。通过Blue Native电泳鉴定出一个复合物，该复合物包括一个带注释的fe氧化还原酶和Etf的β亚基，以及一个膜结合的fe氢化酶的两个亚基。它可能涉及一个甲基萘醌q-循环或离子泵，将不利的质子与NADH还原为氢与有利的克罗丁基辅酶a与NADH还原为丁基辅酶a耦合。本研究旨在对沃尔夫菌中丁酸盐诱导的膜复合物进行厌氧净化和表征。这包括野生型蛋白的增溶，通过几个色谱步骤的纯化，分子和动力学性质的表征。其中一个主要的问题是回答是什么机制导致不利的氢气生产。因此，将使用包括分岔反应在内的联合酶测定法。这项研究的结果可能会扩展我们对通过协同生物的膜复合物进行氧化还原反应耦合的复杂机制的了解，这些反应依赖于氢等化合物的产生，这些化合物在能量有限的环境中作为其协同伙伴的底物。

Syntrophic growth plays an essential role in the recycling of organic matter in anaerobic environments. A distinctive feature of syntrophic metabolism is the need for reverse electron transfer due to the unfavorable thermodynamics of critical oxidation-reduction reactions. Recent genomic and proteomic studies in the obligately anaerobic model organisms Syntrophomonas wolfei and Syntrophus aciditrophicus prvided evidence for an Etf-linked hydrogenase induced during syntrophic growth on butyrate. By Blue Native electrophoresis a complex was identified that comprised an annotated FeS oxidoreductase along with the beta subunit of Etf, and two subunits of a membrane-bound, Fe-hydrogenase. It could involve a menaquinone q-cycle or ion pumps in the coupling of the unfavorable reduction of protons to hydrogen with NADH to the favorable crotonyl-CoA reduction to butyryl-CoA with NADH.This study aims at the anaerobic purification and characterization of the butyrate-induced membrane complex from S. wolfei. This includes solubilization of wild type protein, purification via several chromatographic steps, characterization of the molecular and kinetic properties. One of the major concerns is to answer the questions which mechanism drives the unfavorable hydrogen production. Therefore, combined enzyme assays including bifurcation reactions will be used. The results of this study may extend our knowledge about sophisticated mechanisms involved in the coupling of redox reactions via membrane complexes of syntrophic organisms that depend on the production of compounds such as hydrogen that serve as substrate for their syntrophic partners in an energy limited environment.