Structure-function relationships in metalloenzymes with multiple redox-active centers

智力优势：目前正在研究两种含金属的多血红素呼吸酶，它们参与氨和亚硝酸盐的相互转化，这是生物氮循环的一个重要阶段。来自同一希瓦氏菌的细胞色素c亚硝酸盐还原酶在厌氧呼吸过程中催化亚硝酸盐的六电子还原为氨。来自欧洲亚硝化单胞菌的羟胺氧化还原酶催化羟胺四电子氧化为亚硝酸盐，这是在有氧呼吸过程中氨被氧化为亚硝酸盐的更大过程的一部分。在非生理条件下，与细胞色素c亚硝酸盐还原酶类似，羟胺氧化还原酶也能将亚硝酸盐还原为氨，而细胞色素c亚硝酸盐还原酶也能将羟胺氧化为亚硝酸盐。该项目的主要目的是确定具有大致相似结构的蛋白质是如何被调整以优先地在一个方向或另一个方向引导氨-亚硝酸盐相互转化的。目前的研究重点是ccNiR多电子还原亚硝酸盐生成氨的机理。在细胞色素c亚硝酸盐还原酶催化的反应过程中，中间态的形成和衰变正在使用各种光谱、电化学和结构技术进行研究，其中最著名的是劳厄x射线晶体学和劳厄时间分辨晶体学。这些相对较新的技术具有巨大的未开发潜力。这些技术的开发是项目的重要补充部分。研究结果将提供一个更好的理解众所周知的复杂的氧化还原化学氮在酶的金属中心。更广泛的影响：该项目的高度跨学科性质为研究培训的学生提供了广泛的技能，这将使他们在毕业后从事独立职业时具有很强的竞争力。该项目将涉及平均每年三名本科生研究人员，此外还有两名高中生和两名高中教师将进行夏季研究。受资助的研究生也将直接指导许多本科生和一名高中学生和教师。该项目还提供了对氮循环失衡日益严重的环境问题的见解。氨（肥料的主要成分）和亚硝酸盐是“活性氮”的两个例子；也就是说，许多生物都可以利用氮，而不是“元素氮”，它占我们呼吸的空气的78%，但只有少数细菌可以直接利用。在过去的50年里，随着越来越多的肥料被用于生产粮食和（最近的）生物燃料，活性氮和元素氮之间的平衡已经明显转向了前者。这种转变正在产生许多意想不到的负面后果，这些后果很快就必须得到缓解。更好地了解氨-亚硝酸盐的相互转化将导致更有效地利用氨肥，从而有助于纠正不平衡。

Intellectual Merit:Two metal-containing, multi-heme respiratory enzymes involved in the interconversion of ammonia and nitrite, an important stage in the biological nitrogen cycle, are being investigated. Cytochrome c nitrite reductase from Shewanella oneidensis catalyzes the six-electron reduction of nitrite to ammonia during anaerobic respiration. Hydroxylamine oxidoreductase from nitrosomonas europaea catalyzes the four-electron oxidation of hydroxylamine to nitrite, as part of a larger process in which ammonia is oxidized to nitrite during aerobic respiration. Under non-physiological conditions, hydroxylamine oxidoreductase can also reduce nitrite to ammonia similar to cytochrome c nitrite reductase, while cytochrome c nitrite reductase can oxidize hydroxylamine to nitrite. The primary aim of the project is to determine how the proteins, which have broadly similar architectures, are tailored to shepherd the ammonia-nitrite interconversion preferentially in one direction or the other. The immediate focus is on the mechanism of the multi-electron reduction of nitrite to ammonia by ccNiR. Intermediate states that form and decay during the reactions catalyzed by cytochrome c nitrite reductase are being investigated with the use of a variety of spectroscopic, electrochemical and structural techniques, most notably Laue X-ray crystallography and Laue-based time-resolved crystallography. These relatively new techniques have enormous untapped potential. The development of these techniques is an important complementary part of the project. The research outcome will provide a better understanding of the notoriously complicated redox chemistry of nitrogen at enzymatic metal centers. Broader impacts:The project's highly interdisciplinary nature provides the students in research training with a wide breadth of skills that would make them very competitive when they go on to independent careers after graduation. The project will involve an average of three undergraduate researchers per year, in addition to two high school students and two high school teachers who will perform summer research. The graduate students funded to work on this project will also provide direct supervision for many of the undergraduates and one high school student and teacher. The project also provides insight into the growing environmental problem of nitrogen cycle imbalance. Ammonia (a major component of fertilizer) and nitrite are two examples of "reactive nitrogen;" that is, nitrogen usable by many living organisms, as opposed to "elemental nitrogen" which makes up 78% of the air we breathe, but is directly usable by only a few bacteria. Over the last 50 years the balance between reactive and elemental nitrogen has shifted significantly towards the former, as more fertilizer was generated to produce food and (recently) biofuels. This shift is having many unintended negative consequences, which will soon have to be mitigated. A better understanding of ammonia-nitrite interconversion would lead to the more efficient use of ammonia fertilizer, and thus help redress the imbalance.