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，A教授。威斯康星大学密尔沃基分校的安德鲁帕切科研究氨和亚硝酸盐之间的生物相互转化化学。 氨是肥料的主要成分，亚硝酸盐是“活性氮”的两个例子，即许多生物体都可以使用的氮，而“元素氮”占我们呼吸的空气的78%，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。 这种转变带来了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更有效地利用氨肥料，从而有助于纠正不平衡。 该项目的高度跨学科性质将为研究生，本科生和高中学生提供广泛的技能，使他们在独立的职业生涯中非常有竞争力。 这些高中生将通过美国化学学会经济困难项目的暑期经验招募。 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，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;在没有ccNiR的情况下，这个过程对于细菌的生存来说太慢了。 帕切科小组以前的研究表明，当弱还原剂用作电子源时，可以捕获和研究推定的催化中间体。 该策略将用于捕获亚硝酸盐负载的ccNiR的推定催化中间体，其中活性位点相对于酶的静息状态是1-电子、2-电子和4-电子还原的。 使用UV/维斯停流的动力学研究将用于确定中间体累积的时间尺度，之后将通过快速冷冻淬灭制备这些中间体的样品，并使用各种电子顺磁共振光谱方法（CW-EPR和脉冲-EPR）和穆斯堡尔光谱进行研究。 这些研究的主要目标是确定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、一氧化二氮或羟胺。该奖项反映了NSF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.