Energy conservation in methanogenic archaea associated with the human gut

人类微生物组相关的甲烷古菌在疾病和健康中的可能参与值得进一步研究，以了解它们的生理和代谢。因此，本提案的主要目的是阐明在人类肠道中发现的仅以H2和甲醇或甲胺为底物的产甲烷生物的能量守恒机制。实验将以模式生物发光甲烷发酵球菌（M.）为基础。这种生物不符合目前关于产甲烷菌如何从它们的底物中获得能量的方案。M. luminyensis含有氢营养产甲烷菌中常见的氢化酶/异二硫氧化还原酶复合物（HdrABC/MvhADG），但缺乏节约能量的甲基- h4mpt辅酶M甲基转移酶复合物。那么问题来了，能量是如何在这个有机体中保存的。在之前的资助期内，我们可以证明可溶性HdrABC/MvhADG复合物在发光分枝杆菌的细胞质室中高度活跃。在反应过程中，铁氧还蛋白起电子受体的作用。此外，我们可以证明，在重组HdrD存在的情况下，洗涤的细胞质膜使用还原的铁氧还蛋白（Fdred）来还原异二硫CoM-S-S-CoB。我们目前的假设是Fdred是被F420H2脱氢酶的截断形式（无头Fpo复合物）再氧化的，它错过了F420H2氧化亚基FpoF。因此，电子被转移到直接与无头Fpo复合物相互作用的HdrD上。反过来，HdrD催化CoM-S-S-CoB的还原。整个过程可以耦合到离子移位（H+或Na+）和通过A1A0型ATP合酶合成ATP的电化学离子梯度的产生。为了验证这一假设，计划分析HdrD和fdrd与无头Fpo配合物的相互作用。该方法包括部分纯化Fpo配合物或Fpo亚配合物以重建厌氧电子传递链。此外，还将分析蛋白质与蛋白质之间的相互作用，以阐明电子的路径，并确定电子从Fdred转移到CoM-S-S-CoB的亚基(s)，作为提出的新型节能电子传递链的最终电子受体。

The possible involvement of human microbiome-associated methanoarchaea in disease and health warrants further investigation to understand their physiology and metabolism. Therefore, the major aim of this proposal is to elucidate the energy conservation mechanism in methanogenic organisms found in the human gut which exclusively use H2 and methanol or methylamines as substrates. The experiments will be based on the model organism Methanomassiliicoccus (M.) luminyensis. This organism does not fit into the current scheme of how methanogens gain energy from their substrates. M. luminyensis contains the typical hydrogenase/ heterodisulfide oxidoreductase complex (HdrABC/MvhADG) as found in hydrogenotrophic methanogens but the energy conserving methyl-H4MPT coenzyme M methyltransferase complex is absent. So the question arises how energy is conserved in this organism. In the previous funding period we could show that the soluble HdrABC/MvhADG complex was highly active in the cytoplasmic compartment of M. luminyensis. In the course of the reaction ferredoxin functions as electron acceptor. Furthermore, we could show that reduced ferredoxin (Fdred) is used by washed cytoplasmic membranes to reduce the heterodisulfide CoM-S-S-CoB in the presence of recombinant HdrD. Our current hypothesis is that Fdred is reoxidized by a truncated form of the F420H2 dehydrogenase (headless Fpo complex) which misses the F420H2-oxidizing subunit FpoF. Accordingly, electrons are transferred to HdrD that directly interacts with the headless Fpo complex. In turn, HdrD catalyzes the reduction of CoM-S-S-CoB. The overall process could be coupled to ion translocation (H+ or Na+) and the generation of an electrochemical ion gradient for the synthesis of ATP via a A1A0 type ATP synthase. To verify this hypothesis, it is planned to analyze the interaction of HdrD and Fdred with the headless Fpo complex. The approach includes the partial purification of the Fpo complex or Fpo subcomplexes to reconstitute the anaerobic electron transport chain. Also protein: protein interaction will be analyzed to elucidate the route of electrons and to identify the subunit(s) which are involved in the electron transfer from Fdred to CoM-S-S-CoB as final electron acceptor of the proposed novel energy-conserving electron transport chain in M. luminyensis.