UNDERSTANDING THE MOLECULAR DETAILS OF BIOLOGICAL METHANE FORMATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 厌氧产甲烷古菌(产甲烷菌)形成甲烷作为其新陈代谢的最终产物。据估计，产甲烷菌每年产生约10亿吨甲烷。甲烷是一种强有力的温室气体，但也是一种潜在的有价值的可再生能源。这把？双刃剑？使生物甲烷产生的研究与我们当今世界面临的两个最关键的挑战相关。甲基辅酶M还原酶(MCR)存在于所有产甲烷菌中，催化生物产生甲烷的最后一步。甲基辅酶M(甲基-SCoM，2-(甲硫基)乙磺酸盐)和辅酶B(CoBSH，N-7-巯基庚酰基苏氨酸磷酸)是反应的底物，其中CoBSH是甲基-SCoM 2电子还原生成甲烷和CoBS-SCoM混合二硫化物的电子供体。MCR含有辅酶F430，这是一种氧化还原活性的四氢珊瑚镍，是自然界中已知的还原程度最高的四吡咯。辅酶F430的巨大还原潜力意味着稳定该酶的还原和活性形式用于结构研究到目前为止是不可能的。到目前为止，结晶学研究一直是以氧化的催化非活性形式进行的。在与密歇根大学医学院Steve Ragsdale的合作中，我们现在相信，通过保持从微生物生长到晶体冻结的厌氧条件，我们可以产生活性形式的酶MCRred1，在单晶中的占有率为70%。有了这些晶体，大量冷冻捕获的中间体和被抑制的形式就变得容易获得。晶体的高衍射质量(高达1.2埃分辨率)使我们能够了解结构与这种非常不寻常的酶的功能之间的关系。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The anaerobic methanogenic archaea (methanogens) form methane as an end product of their metabolism. It has been estimated that methanogens produce approximately 1 billion tons of methane every year. Methane is a potent greenhouse gas, but also represents a potential valuable source of renewable energy. This ?double-edged sword? makes the study of biological methane generation of relevance to two of the most critical challenges we face in the World today. Found in all methanogens, methyl-coenzyme M reductase (MCR) catalyzes the final step in biological methane production. Methyl-coenzyme M (methyl-SCoM, 2-(methylthio)ethanesulfonate) and coenzyme B (CoBSH, N-7-mercaptoheptanoylthreonine phosphate) are the substrates for the reaction, in which CoBSH serves as the electron donor for the 2 electron reduction of methyl-SCoM to produce methane and a CoBS-SCoM mixed disulfide. MCR contains coenzyme F430, a redox active nickel tetrahydrocorphin that is the most reduced tetrapyrrole known in nature. The large reduction potential of coenzyme F430 means that stabilizing the reduced and active form of the enzyme for structural studies has proved impossible up to this point. Crystallographic studies thus far have been conducted with oxidized catalytically inactive forms. In collaboration with Steve Ragsdale (University of Michigan Medical School) we now believe we can generate the active form of the enzyme, MCRred1, at ~70% occupancy in single crystals through maintaining anaerobic conditions from microbe grow-up to crystal freezing. With these crystals in hand, a plethora of freeze trapped intermediates and inhibited forms become accessible. The high diffraction quality of the crystals (up to 1.2 Angstroms resolution) allows us to understand how structure relates to function in this highly unusual enzyme.