Differentiating the Two Complementary Flavins in a Bifurcating Electron Transfer Flavoprotein

在化学系生命过程化学计划的支持下，肯塔基大学的安妮-弗朗西斯·米勒教授和她的团队正在研究细菌是如何设法优化其能量效率的。科学已经发展到可以从风和阳光中提供人类所需的所有能源的地步。然而，这些都是间歇性的低密度来源，需要加大力度为吹风机等家用电器供电。事实证明，细菌拥有能够从现成的燃料来源产生集中能量的酶。这些酶采用了一种机制，即从廉价而充足的燃料中获得两个电子，但只允许其中一个电子“下坡”运行。这种酶以某种方式利用这一有利的过程来驱动一个不利的“上坡”反应，从而产生比起始电子载体更强大的电子载体。为了使材料的设计能够做到这一点，拟议中的研究试图了解大自然是如何做到这一点的。与维生素核黄素相关的黄素分子作为辅因子结合在蛋白质中，是这一机制的核心。这项计划中的研究试图阐明这种蛋白质如何调节黄素的反应性。黄素是黄色的，属于更大的色素化学类别。这项工作的更广泛的影响将是为非化学家开发一门课程，该课程将利用纤维艺术和染色活动来解释核心化学概念。这种实践学习方法旨在接触到那些不认为基于书本和讲座的课程具有说服力的受众。因此，拟议的工作旨在吸引更多的受众参与化学的乐趣和好奇心，并利用化学为社会提供更大的服务。本研究试图了解分叉电子转移黄素蛋白(ETF)如何调节其两个黄素的反应性，使一个黄素仅执行单电子转移，而另一个黄素具有非常高的能量半喹酮状态，导致一个电子的转移与第二个电子的转移紧密耦合。一种特殊的假设是，黄素腺嘌呤二核苷酸(FAD)中的焦磷酸基团可能具有调节黄素活性的作用。拟议的工作还涉及ETF的一种新状态所固有的性质和经验教训，该状态被提议涉及两种黄素的合作。这项研究综合了计算、光谱、电化学和生化策略。为了开发计算方法并能够从潜在的电子结构和反应性方面解释黄素光谱，提出了一种系统的方法，从共价修饰的黄素开始。一旦得到验证，计算方法将被用来推断黄素结合的不同蛋白质位点产生的电子扰动。虽然黄素光谱学是拟议研究的中心，但该提案更广泛地利用颜料作为工具，使非科学家受众能够接触到化学的核心概念。关于纤维艺术中的植物颜料的实验和讲座课程将发展为一个模块化的在线课程，包括实践活动的指导。该小组将组装行刑工具包，农村学校或在家上学的孩子将能够借到，以便跟随录制的示范和演示。这种方法寻求克服阻碍许多人从事化学工作的障碍。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

With support from the Chemistry of Life Processes Program in the Division of Chemistry, Professor Anne-Frances Miller and her team at the University of Kentucky are examining how bacteria have managed to optimize their energy efficiency. Science has advanced to the point that it is now possible to supply all the energy needed by mankind, from wind and sunlight. However, these are intermittent, low-density sources that need to be 'stepped up' to power appliances such as hair driers. It turns out that bacteria possess enzymes capable of producing concentrated power from readily available fuel sources. These enzymes employ a mechanism wherein pairs of electrons are obtained from a cheap abundant fuel, but only one of the electrons is allowed to run 'down-hill'. The enzyme somehow harnesses this favorable process to drive an unfavorable 'up-hill' reaction that yields a much more potent electron carrier than the starting one. To enable design of materials able to do the same, the proposed research seeks to learn how nature does this. Flavin molecules related to the vitamin riboflavin, bound in proteins as cofactors are central to the mechanism. The planned research seeks to elucidate how the protein adjusts the flavin's reactivity. Flavins are yellow and fall into the larger chemical category of pigments. The broader impact of this work will be to develop a course for non-chemists that will employ fiber art and dyeing activities to explain core chemical concepts. This hands-on learning approach is designed to reach audiences who do not find book- and lecture-based courses compelling. Thus, the proposed work aims to engage a larger audience in the fun and curiosity of chemistry, and to harness chemistry in larger service to society.This research seeks to learn how bifurcating electron transfer flavoproteins (ETFs) tune the reactivities of their two flavins to cause one flavin to execute single electron transfers only, but the other flavin to have a very high energy semiquinone state that causes transfer of one electron to be tightly coupled to transfer of the second. A specific hypothesis is that the pyrophosphate group built into flavin adenine dinucleotide (FAD) could have agency in modulating the activity of the flavin. The proposed work also addresses the nature and lessons inherent in a novel state of the ETF that is proposed to involve cooperation of both flavins. The research integrates computational, spectroscopic, electrochemical, and biochemical strategies. To develop computational approaches and enable interpretation of flavin optical spectra in terms of underlying electronic structure and reactivity, a systematic approach is proposed, beginning with covalently modified flavins. Once validated, computational approaches would then be employed to infer the electronic perturbations produced by different protein sites in which flavins are bound. While flavin spectroscopy is at the center of the proposed research, the proposal exploits pigments more generally as vehicles to make core concepts of chemistry accessible to non-scientist audiences. A lab and lecture course on plant pigments in fiber arts will be developed into a modular on-line course, including guidance for hands-on activities. The team will assemble execution kits that rural schools or homeschoolers will be able to borrow, in order to follow along with recorded demonstrations and presentations. This approach seeks to overcome barriers that prevent many people from engaging with chemistry.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.