Structure-Function Relationships in Metalloenzymes with Multiple Redox-active Centers

智力MeritResearch将对三种含金属的蛋白质进行研究，这些蛋白质允许特定的细菌相互转化氨和亚硝酸盐。 如果没有这些蛋白质，这种相互转化对于细菌的生存来说太慢了。 该项目涉及时间分辨X射线晶体学和劳厄晶体学方法的开发和应用，以获得三种多血红素呼吸酶的结构信息;细胞色素c腈还原酶，octaheme tetrahydrophosphate还原酶和羟胺氧化还原酶，这些酶具有广泛相似的结构。该项目的目标是阐明铵和亚硝酸盐的多电子相互转化的机制。调查技术相对较新，具有巨大的未开发潜力，开发这些技术是该项目的一个重要补充目标。 在传统晶体学的数据收集过程中，整个晶体暴露在X射线下几个小时。 然而，将蛋白质暴露于X射线通常会导致常规实验时间尺度的变化。这对于含金属的蛋白质尤其如此，例如本项目中感兴趣的蛋白质。因此，获得的结构往往不是原始分子的结构，而是反映原始结构和X射线修饰后蛋白质结构的平均值。相比之下，劳厄图案在100皮秒内获得，对于辐射损伤来说太短了。 因此，劳厄方法非常适合于对X射线损伤敏感的蛋白质的研究。 劳厄方法也可以用来制作反应过程中分子变化的“电影”，通过将反应开始后不同时间的曝光拼接在一起（时间分辨晶体学）。更广泛的影响每年有三名研究生将参与这个项目。 该项目的高度跨学科性质为学生提供了广泛的技能，这将使他们在毕业后从事独立职业时具有很强的竞争力。 该项目将赞助平均每年三名本科研究人员，除了两名高中生和两名高中教师谁将进行夏季研究。 该项目支持的研究生还为许多本科生以及一名高中生和教师提供直接监督。 该项目还提供了对氮循环不平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，许多生物体都可以使用的氮，而不是“元素氮”，它占我们呼吸的空气的89%，但只有少数细菌可以直接使用。 在过去的50年里，活性氮和元素氮之间的平衡已经显著地向前者转移，因为更多的肥料被用来生产粮食和（最近）生物燃料。 这种平衡的转变正在产生许多意想不到的负面后果，这些后果很快就会得到缓解。 更好地了解氨-亚硝酸盐的相互转化可能会导致氨肥料的更有效利用，从而有助于纠正不平衡。

Intellectual MeritResearch will be undertaken on three metal-containing proteins that allow particular bacteria to interconvert ammonia and nitrite. Without these proteins, such an interconversion would be too slow for bacterial survival. This project involves the development and application of time-resolved X-ray crystallography and Laue crystallography methods in an effort to obtain structural information on three multi-heme respiratory enzymes; cytochrome c nitrile reductase, octaheme tetrahtionate reductase and hydroxylamine oxidoreductase, enzymes which have broadly similar architectures. The goal of the project is to elucidate the mechanism of the multi-electron interconversion of ammonium and nitrite. The investigative techniques are relatively new and have enormous untapped potential, the development of which is an important complementary aim of the project. During data collection in conventional crystallography the entire crystal is exposed to X-rays for several hours. However, exposing a protein to X-rays often results in changes on the time scale of the conventional experiment. This is especially true for metal-containing proteins such as the ones of interest in this project. As a result, the structure one obtains is often not that of the original molecule, reflecting instead the average of the original structure and those of the protein after modification by the X-rays. By contrast, a Laue pattern is obtained in 100 picoseconds, too short a time for radiation damage to appear. As a consequence, the Laue method is ideally suited to the investigation of proteins that are susceptible to X-ray damage. The Laue method can also be used to make a "movie" of molecular changes during a reaction, by piecing together exposures taken at varying times after reaction initiation (time resolved crystallography). Broader impactThree graduate students per year will work on this project. The project's highly interdisciplinary nature provides the students with a wide breadth of skills that will make them highly competitive when they go on to independent careers after graduation. The project will sponsor 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 supported on this project also provide direct supervision for many of the undergraduates as well as 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 89% 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 in balance is having many unintended negative consequences, which will soon have to be mitigated. A better understanding of ammonia-nitrite interconversion may lead to the more efficient use of ammonia fertilizer, and thus help redress the imbalance.This project is receiving co-funding from the Chemistry of Life Processes program in the Chemistry Division