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

在陆地和海洋生态系统中，植物的生长往往受到基本元素氮的可用性的限制。生物固氮是将氮气转化为植物可利用的铵的反应，是控制地球上生态系统肥力的关键反应。固氮酶是负责这一反应的酶(生物催化剂)，以三种不同的形式存在，称为异构体。每种异构体都有不同的特性，包括不同的效率、微量金属需求和最适温度。然而，它们对环境中固氮的相对贡献目前尚不清楚。本项目旨在使用一种新开发的方法来提高我们对这三种固氮酶亚型的分布和活性的了解。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)，以及这些服务可能如何演变到未来。它还将为本科生和研究生提供重要的教育培训，并为K-5学生的科学活动提供材料，重点是土壤养分的来源及其对植物生长的作用。固氮酶的三种同工型是钼固氮酶，以及仅含V和Fe的固氮酶(有时被称为“替代”固氮酶)。替代固氮酶的活性可以用一种新的方法(ISARA，用于同位素乙炔还原试验)来区分，该方法是经典的乙炔还原试验(ARA)的变体。样品与乙炔孵育，随着时间的推移测量乙烯的产生，以估计固氮酶的活性。乙炔还原反应的~(13)C同位素分馏有助于判断是钼固氮酶还是替代固氮酶进行了该反应。拟议的项目有两个部分：1-确定三种固氮酶同工酶在沿海海洋和北方生态系统中的贡献；2--替代固氮的实验室调查，特别侧重于描绘金属、温度和细胞生理控制对同工酶活性的相互作用。这项工作将为替代固氮酶在生态系统固氮中的贡献提供定量数据。这也将为替代固氮的控制提供新的线索。如果确认了替代固氮的重要性，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的氮循环的理解。

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.