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

在化学系生命过程化学项目的支持下，威斯康星大学密尔沃基分校的A. Andrew Pacheco教授研究氨和亚硝酸盐之间生物相互转化的化学过程。氨是肥料的主要成分，亚硝酸盐是“活性氮”的两个例子，即许多生物可以利用的氮，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。这种转变带来了许多意想不到的负面后果，这些后果很快就必须得到缓解。更好地了解氨与亚硝酸盐的相互转化，可能会导致更有效地利用氨肥，从而有助于纠正不平衡。该项目的高度跨学科性质将为从事研究的研究生、本科生和高中生提供广泛的技能，使他们在独立的职业生涯中具有很强的竞争力。这些高中生将通过美国化学会经济弱势群体项目的暑期体验项目被招募。拟议的研究将集中在细胞色素-c亚硝酸盐还原酶（ccNIR）的反应机制上，这种酶可以使某些细菌将亚硝酸盐还原为氨。细菌可以从这个过程中提取能量；在没有ccNiR的情况下，这个过程对于细菌的生存来说太慢了。Pacheco小组先前的研究表明，当弱还原剂用作电子源时，可以捕获和研究假定的催化中间体。该策略将用于捕获亚硝酸盐负载ccNiR的推定催化中间体，其中活性位点是相对于酶的静息状态减少的1电子，2电子和4电子。使用UV/Vis停止流动的动力学研究将用于确定中间体积累的时间尺度，之后这些中间体的样品将通过快速冷冻淬火制备，并使用各种电子顺磁共振波谱方法（w - epr和脉冲epr）和穆斯堡尔波谱进行研究。这些研究的主要目的是确定ccNiR活性位点如何优化以抵抗在催化过程中释放部分还原的含氮中间体，如一氧化氮、氧化亚氮或羟胺。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes program in the Division of Chemistry, Professor A. Andrew Pacheco of the University of Wisconsin-Milwaukee studies the chemistry of biological interconversion between ammonia and nitrite. Ammonia, which is a major component of fertilizers, 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 has 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. The project’s highly interdisciplinary nature will provide the graduate, undergraduate and high school students who conduct the research with a wide breadth of skills that will make them very competitive in their independent careers. The high school students will be recruited through Summer Experiences for the Economically Disadvantaged Program of the American Chemical Society. The proposed research will concentrate 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; in the absence of ccNiR, this process would be too slow for bacterial survival. Previous studies by the Pacheco group showed that putative catalytic intermediates can be trapped and studied when weak reductants are used as electron sources. This strategy will be used to trap putative catalytic intermediates of nitrite-loaded ccNiR in which the active site is 1-electron, 2-electron and 4-electron reduced relative to the resting state of the enzyme. Kinetic studies using UV/Vis stopped-flow will be used to determine the timescales on which intermediates accumulate, after which samples of these intermediates will be prepared by rapid freeze-quench and investigated using a variety of electron paramagnetic resonance spectroscopic methods (CW-EPR and pulsed-EPR) and Mossbauer spectroscopy. The primary goal of these studies is to determine how the ccNiR active site is optimized to resist releasing partially-reduced nitrogenous intermediates, such as nitric oxide, nitrous oxide or hydroxylamine, during catalysis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.