Exploration of the chemical and mechanistic diversity of mixed-valent iron oxygenases and oxidases

在化学系生命科学化学项目的支持下，宾夕法尼亚州立大学的Carsten Krebs、J. Martin Bollinger、Jr.和Alexey Silakov教授将使用分析、动力学和光谱方法来了解一种新的功能类铁酶如何利用分子氧（O2）在细菌的营养获取和生物合成途径中进行复杂的生化转化。这种酶催化的反应通常超出了合成化学家目前的能力，可以直接使用或有目的地进化，以制造包括药物在内的高价值化学品。大多数这些酶都有由一个或两个还原铁（II）形式的铁离子组成的辅因子。2006年，研究人员发现了一种新的功能类酶，它使用混合价双铁辅助因子，即含有一个还原铁（II）和一个氧化铁（III）离子的辅助因子来催化不同底物的复合四电子氧化。混合价二铁酶的第一个亚家族是如何催化它们的反应的，目前还只是部分了解。该研究将通过对反应性中间体的直接结构询问来揭示酶的催化循环的关键原理。科学家们还将与合作者一起研究具有“TIM-barrel”结构的多核铁酶。这些酶催化肽的显著重排，以制造铜伴侣和抗生素，也使用混合价二铁或三铁辅助因子。该项目将是宾夕法尼亚州立大学集团两年一次的生物无机化学培训计划中揭示金属生物化学一般原理和方法的几个项目之一，该计划通过录制和公开的讲座和实践培训课程来培训来自世界各地的本科生和博士生，涉及20多种生物无机化学和相关研究领域的核心技术。在该项目的第一个目标中，将通过同位素标记和脉冲电子顺磁共振（EPR）方法探索相关的双铁(III)-超氧化物中间体的性质，这些中间体在三种不同的酶中提取氢原子以引发氧化碳-碳和碳-磷键裂解反应。追踪氧原子从O2和H2O进入产物的路径的分析方法将有助于解释令人困惑的初步观察结果并阐明其机制。在第二个目标中，新的异二聚体TIM-barrel混合价铁酶的辅因子的性质将通过Mössbauer和EPR光谱来定义。关键的问题是，辅因子中是否有两个或三个铁离子，以及它们是否形成超氧- fe2 -3络合物，这些络合物抽出氢原子，引发含半胱氨酸肽的复杂重排，从而产生被称为甲烷菌和抗生素l -硫谷氨酸的铜伴侣。从这些研究中产生的理解将为新的生物催化反应的发现和指导进化提供信息。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 Sciences Program in the Division of Chemistry, Professors Carsten Krebs, J. Martin Bollinger, Jr., and Alexey Silakov at Penn State University will use analytical, kinetic, and spectroscopic methods to understand how a new functional class of iron enzymes use molecular oxygen (O2) to carry out complex biochemical transformations in nutrient-acquisition and biosynthetic pathways in bacteria. Such enzymes catalyze reactions that generally exceed the current capabilities of synthetic chemists and could be used directly or purposefully evolved to make high-value chemicals including drugs. Most of these enzymes have cofactors consisting of one or two iron ions in the reduced iron(II) form. In 2006 the investigators identified a new functional class of enzymes that use mixed-valent diiron cofactors, i.e. cofactors that contain one reduced iron(II) and one oxidized iron(III) ion to catalyze complex four-electron oxidations of diverse substrates. How this first subfamily of mixed-valent diiron enzymes catalyze their reactions is only partly understood. The research will seek to reveal key principles of the catalytic cycles of the enzymes by direct structural interrogation of reactive intermediates. Together with collaborators, the scientists will also study multinuclear iron enzymes that have an architecture called "TIM-barrel." These enzymes catalyze remarkable rearrangement of peptides to make copper chaperones and antibiotics and also use mixed-valent di- or tri-iron cofactors. The project will be one of several used to reveal general principles and approaches in metallobiochemistry within the Penn State group's biennial bioinorganic-chemistry training program, which trains undergraduate and Ph.D. students from around the world via recorded and publicly available lectures and hands-on practical training sessions on more than 20 techniques central for bioinorganic chemistry and related research areas.In the first aim of the project, the nature of the related diiron(III)-superoxide intermediates that abstract hydrogen atoms to initiate oxidative carbon-carbon and carbon-phosphorus bond cleavage reactions in three different enzymes will be probed by isotope-labeling and pulse electron paramagnetic resonance (EPR) methods. Analytical methods to trace the path of the oxygen atoms from O2 and H2O into the products will help explain puzzling preliminary observations and clarify their mechanisms. In the second aim, the nature of the cofactors in the new heterodimeric TIM-barrel, mixed-valent iron enzymes will be defined by Mössbauer and EPR spectroscopies. The crucial questions are whether there are two or three iron ions in the cofactors and whether they form superoxo-Fe2-3 complexes that abstract hydrogen atoms to initiate the complex rearrangements of cysteine-containing peptides that make the copper chaperones known as methanobactins and the antibiotic L-thiaglutamate. The understanding that will emerge from these studies will inform discovery and directed evolution of new biocatalytic reactions.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.