ALTERNATIVE NITROGENASES: HOW MUCH DO THEY CONTRIBUTE TO N2 FIXATION AND WHY?

在陆地和海洋生态系统中，植物生长往往受到必需元素氮的可用性的限制。生物固氮是一种将氮气转化为植物可利用铵的反应，是控制地球生态系统肥力的关键反应。负责该反应的酶（生物催化剂）氮酶以三种不同的形式存在，称为同种异构体。每种异构体都具有独特的性质，包括不同的效率、痕量金属需求和最佳温度。然而，它们对环境中固氮的相对贡献目前尚不清楚。本项目旨在利用一种新开发的方法来提高我们对三种氮酶同工型的分布和活性的认识。该项目将更好地了解固氮为人类社会提供的服务（如生态系统肥力的变化）以及它们如何演变到未来。它还将为本科生和研究生提供重要的教育培训，并为K-5学生提供科学活动的材料，重点是土壤养分的来源及其对植物生长的作用。氮酶的三种同工型分别是钼氮酶、纯钒和纯铁氮酶（有时被称为“替代”氮酶）。使用一种新的方法（ISARA，即同位素乙炔还原法）可以将替代氮酶的活性与mo -氮酶的活性区分开来，该方法是对经典乙炔还原法（ARA）的一种改进。样品与乙炔孵育，并测量乙烯产量随时间的变化，以估计氮酶活性。乙炔还原反应的13C同位素分馏可用于诊断是Mo-或替代氮素酶进行反应。拟议的项目有两个组成部分：1-确定三种氮酶亚型在沿海海洋和北方生态系统中的贡献；2-另类固氮的实验室研究，特别侧重于描述金属、温度和细胞生理控制对异构体活性的相互作用。本研究将为研究替代氮酶对生态系统固氮的贡献提供定量数据。它也将揭示新的控制替代固氮。如果替代固氮的重要性得到证实，可能需要重新检查基于同位素和ara的对局部和全球尺度上氮循环的理解。

In terrestrial and marine ecosystems, plant growth is often limited by the availability of the essential element nitrogen. Biological nitrogen fixation, the reaction that transforms nitrogen gas into plant-available ammonium, is a key reaction that controls the fertility of ecosystems on the planet. Nitrogenase, the enzyme (biological catalyst) responsible for this reaction, exists in three different forms, called isoforms. Each isoform has characteristic properties, including different efficiencies, trace metal requirements and optimum temperatures. Yet, their relative contributions to nitrogen fixation in the environment are currently unknown. This project aims at using a newly developed method to improve our understanding of the distribution and activity of the three nitrogenase isoforms. This project will provide a better understanding of the services (such as changes in ecosystem fertility) provided by nitrogen fixation to human societies and how they may evolve into the future. It will also provide important educational training to undergraduate and graduate students, and material for science activities with K-5 students focused on the origin of soil nutrients and their role for plant growth.The three isoforms of nitrogenase are the Mo-nitrogenase, and the V- and the Fe-only nitrogenases (sometimes referred to as the "alternative" nitrogenases). The activity of the alternative nitrogenases can be distinguished from that of the Mo-nitrogenase using a new method (ISARA, for Isotopic Acetylene Reduction Assay), which is a variation on the classic Acetylene Reduction Assay (ARA). The samples are incubated with acetylene, and ethylene production is measured over time to estimate nitrogenase activity. The 13C isotope fractionation of acetylene reduction is diagnostic of whether the Mo- or the alternative nitrogenases carry out the reaction. The proposed project has two components: 1- Determination of the contribution of the three nitrogenase isoforms in coastal marine and boreal ecosystems; 2- Laboratory investigations of alternative nitrogen fixation, with particular focus on delineating the interplay of metal, temperature, and cellular physiological controls on isoform activity. This work will provide the quantitative data on the contribution of alternative nitrogenases to ecosystem nitrogen fixation. It will also shed new light on the controls of alternative nitrogen fixation. If the importance of alternative nitrogen fixation is confirmed, it may require a reexamination of isotope- and ARA-based understanding of the nitrogen cycle on local and global scales.