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

这个项目的最终目的是更好地了解生物驱动的氨-亚硝酸盐相互转化的化学。氨(肥料的主要成分)和亚硝酸盐是“活性氮”的两个例子；即许多生物可利用的氮，而不是“元素氮”，后者占我们呼吸的空气的78%，但只有少数细菌能直接利用。在过去的50年里，随着更多的化肥被用于生产粮食和(最近)生物燃料，活性氮和元素氮之间的平衡已经显著转向前者。更好地了解氨-亚硝酸盐的相互转化可能会导致更有效地利用氨肥，从而有助于纠正这种不平衡。每年有一到三名研究生被直接资助参与这个项目。该项目的高度跨学科性质为学生提供了广泛的技能，使他们在毕业后从事独立职业时非常有竞争力。除了研究生，在任何一年里，平均有三名本科生和两名高中生参与该项目。本科生通常通过国家科学基金会的补充助学金或威斯康星大学密尔沃基分校的机构支持来资助，而高中生则通过美国化学学会的“种子计划”项目来资助。资助这个项目的研究生也为更初级的研究人员提供直接指导。目前的研究集中在细胞色素c亚硝酸盐还原酶(CcNIR)的反应机理上，ccNIR是一种允许某些细菌将亚硝酸盐还原为氨的酶。细菌可以从这个过程中提取能量，如果没有ccNiR，细菌的生存将太慢。该项目的长期目标是确定ccNiR和羟胺氧化还原酶(Hao)是如何定制的，以优先在一个方向或另一个方向引导氨-亚硝酸盐的相互转化。羟胺氧化还原酶是一种不同的细菌用来从氨氧化中提取能量的酶，其结构与ccNiR的结构大体相似。在ccNiR催化的反应中形成和衰变的中间态正在使用各种方法进行研究，最著名的是时间分辨结晶学技术。X射线结晶学是确定生物大分子中原子三维排列的为数不多的方法之一。时间分辨方法利用超短高强度X射线脉冲，这些脉冲可在先进光子源的BioCARS或斯坦福大学的LCLS X射线自由电子激光器等设施中获得，以在反应过程中创建酶分子的“定格”快照。通过将反应开始后不同时间拍摄的曝光拼接在一起，就可以得到分子变化的“电影”。这些相对较新的快速结晶学技术具有巨大的未开发潜力，其开发是该项目的一个重要补充目标。

The ultimate aim of this project is to better understand the chemistry of biologically driven ammonia-nitrite inter-conversion. 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. A better understanding of ammonia-nitrite inter-conversion may lead to the more efficient use of ammonia fertilizer, and thus help redress the imbalance. One to three graduate students per year are funded directly to work on this project. The project's highly interdisciplinary nature provides the students with a wide breadth of skills that make them very competitive when they go on to independent careers after graduation. In addition to graduate students, an average of three undergraduate researchers and two high school students normally work on the project during any given year. The undergraduates are typically financed through supplemental grants from NSF, or through institutional support from the University of Wisconsin Milwaukee, while the high school students are funded through the American Chemical Society's "Project SEED" program. The graduate students funded to work on this project also provide direct supervision for the more junior researchers.The current research concentrates on the reaction mechanism of cytochrome-c nitrite reductase (ccNIR), an enzyme that allows certain bacteria to reduce nitrite to ammonia. The bacteria can extract energy from the process, which without ccNiR would be too slow for bacterial survival. The long-term aim of the project is to determine how ccNiR and hydroxylamine oxidoreductase (HAO), an enzyme that different bacteria use to extract energy from ammonia oxidation, and that has an architecture broadly similar to ccNiR's, are tailored to shepherd the ammonia-nitrite inter-conversion preferentially in one direction or the other. Intermediate states that form and decay during the reaction catalyzed by ccNiR are being investigated using a variety of methods, most notably time-resolved crystallographic techniques. X-ray crystallography is one of the few methods available for determining the 3-dimensional arrangement of atoms in a biological macromolecule. Time-resolved methods take advantage of ultrashort high intensity X-ray pulses, which are available at facilities such as BioCARS at the Advanced Photon Source, or the LCLS X-Ray free electron laser at Stanford, to create "freeze-frame" snapshots of enzyme molecules during the course of reactions. By piecing together the exposures taken at varying times after a reaction is initiated, "movies" of molecular changes are obtained. These relatively new fast crystallography techniques have enormous untapped potential, the development of which is an important complementary aim of the project.