Mechanism and Structure of Nitrous Oxide Reductase

本研究主要针对反硝化途径中的关键酶进行生化和结构生物学研究。反硝化作用是全球氮循环的内在组成部分，也是平衡该循环的途径，将固定的氮素返回到大气中。固氮、同化和反硝化作用的环境生物学对农业生产力和水质有很大影响。反硝化作用可能会向大气中释放一氧化二氮(N2O)，从而导致臭氧消耗和全球变暖。因此，关于反硝化途径的生物学和生物化学的基础研究与许多具有重大社会意义的问题之间存在着明确和直接的联系。具体地说，本研究涉及反硝化过程中的关键酶--一氧化二氮还原酶的结构、机理和组装。在绝大多数反硝化菌中，一氧化二氮还原酶是反硝化途径中的终端酶。这项研究将研究一氧化二氮还原酶中新的铜中心的结构和反应活性，以及它们在催化中的作用。将对还原N2O的质子转移和电子转移步骤进行详细的实验研究。还将研究直接与底物反应的完全还原形式的酶的结构。另一种名为NosX的蛋白质似乎也在N2O的减少中发挥了关键作用。假设是，NosX激活了细胞中的还原酶，并再生了已经失活的酶。这一假设将通过几种方法得到彻底检验。一氧化二氮还原酶和一氧化氮合酶的高效表达方法已经被成功地开发出来，并将被用来制备蛋白质以供研究。定点突变，结合光谱和动力学方法，将被用来探索一氧化二氮还原酶和NOX中催化位点的结构、成键和反应活性。蛋白质的结构研究将通过核磁共振(核磁共振)或X射线结晶学进行。本科生和研究生都将直接参与这个项目。该项目的背景和意义具有广泛的公众利益，并将由首席调查员在多个地点介绍和介绍。更广泛的影响。密歇根州立大学化学和生物化学系是NSF本科生研究体验(REU)网站，PI是参与该网站的教职员工之一。REU计划的一个重要目标是为美洲原住民学生和来自其他代表性不足群体的学生提供研究机会。预计将有一到两名本科生(每年)参与这项研究，作为他们参与REU计划的一部分。此外，该项目可能会支持蒙大拿州比林斯市落基山学院的学生参与，这是一所本科生学院，本科生研究机会极其有限。关于反硝化的研究与农业和环境问题有着明确的联系，因此成为非正式介绍和讨论基础研究与对社会的益处之间的关系和联系的有效中心。脱氮途径与环境、农业和能源问题之间的明确联系在与潜在学生、本科生和研究生、社区团体和利益集团的谈话中经常得到强调。这位首席调查员也是密歇根州立大学博兹曼分校(蒙大拿州领先的研究机构，卡内基一所“非常高的研究活动”大学)的教务长兼学术事务副总裁，每年都有许多机会讲述研究和发现对本科生教育和该州经济发展的重要性。他经常会见州政府成员、立法者和许多选区。

This research focuses on the biochemistry and structural biology of the key enzyme in the denitrification pathway. Denitrification is an intrinsic part of the global nitrogen cycle and is the pathway that balances the cycle, returning fixed nitrogen to the atmosphere. The environmental biology of nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release nitrous oxide (N2O) to the atmosphere, thereby contributing to ozone depletion and global warming. Hence, there exist clear and direct linkages between basic research on the biology and biochemistry of the denitrification pathway and numerous issues of substantial societal interest. Specifically, this research is concerned with the structure, mechanism, and assembly of the key enzyme in denitrification, nitrous oxide reductase. In the vast majority of denitrifying organisms, nitrous oxide reductase is the terminal enzyme in the denitrification pathway. The research will investigate the structures and reactivities of the novel copper sites in nitrous oxide reductase, and their roles in catalysis. Detailed experimental studies of proton-transfer and electron-transfer steps in the reduction of N2O will be carried out. The structure of the fully reduced form of the enzyme, which reacts directly with substrate, will also be pursued. An additional protein, named NosX, also appears to play a key role in the reduction of N2O. The hypothesis is that NosX activates the reductase in cells and also regenerates enzyme that has become inactivated. This hypothesis will be thoroughly tested by several approaches. Successful over-expression methods for nitrous oxide reductase and NosX have been developed and will be exploited to prepare proteins for study. Site-directed mutagenesis, together with spectroscopic and kinetics methods will be used to probe the structure, bonding, and reactivity of the catalytic sites in nitrous oxide reductase and NosX. Structural studies of the proteins will be carried out by nuclear magnetic resonance (NMR) or X-ray crystallography. Both undergraduate and graduate students will be directly involved in the project. The context and significance of the project is of broad public interest, and will be presented and featured in multiple venues by the Principal Investigator. Broader Impacts. The Department of Chemistry and Biochemistry at Michigan State University is a NSF Research Experiences for Undergraduates (REU) site, and the PI is a member of the participating faculty. An important aim of the REU program is to provide research opportunities for Native American students and students from other underrepresented groups. It is anticipated that one or two undergraduates (annually) will be involved in this research as part of their participation in the REU program. In addition, this project may support the participation of students from Rocky Mountain College in Billings, Montana, an undergraduate college with extremely limited opportunities for undergraduate research. The research on denitrification, with its clear connections to agriculture and to environmental issues, has served as an effective focal point for informal presentations and discussions on the relationships and linkages between basic research and benefits to society. The clear connections between the denitrification pathway and environmental, agricultural, and energy issues are regularly emphasized in talks to prospective students, undergraduate and graduate students, community groups, and interest groups. The Principal Investigator, who also serves as the Provost and Vice President for Academic Affairs at MSU-Bozeman (Montana's leading research institution and a Carnegie "very high research activity" university), has many opportunities annually to speak to the importance of research and discovery to undergraduate education and to economic development in the state. He meets frequently with members of state government, legislators, and numerous constituencies.