Biosynthesis and Reactivity of the Active Site of the [FeFe]-Hydrogenases

氢酶普遍存在于细菌、古生菌和一些高等生物中。 氢酶存在于许多病原体中，包括一些存在于人体肠道中的病原体。[FeFe]和 [NiFe]-氢酶介导最基本的化学反应：氢与 质子和还原当量。由于氢是一种不同寻常的底物，这些酶在结构上也是 特别是具有一系列独特的辅因子，特别是氢结合和释放的位置。同样， 这些活性部位的生物合成涉及到精细而新颖的生物化学。除了……之外 生物物理方法，这些机制的阐明依赖于有机金属化学，特别是 因为底物(H2、H+、H-)对于传统的生物物理方法通常是不可见的。 本项目旨在阐明[FeFe]酶的生物合成和作用机制，越快 两种主要的氢酶中的一种，以及最容易开发用于其他用途的一种。这个 工作包括合成建议的中间体，光谱和电化学表征， 体外试验和同位素标记。它还依赖于与提供 理论、基于同步加速器的光谱学、自旋共振和体外测试方面的专门技能。 这个项目特别关注最近确认的氮杂硫酸酯(ADT)辅助因子，它是 从机械论的角度来看，这是酶最显著的成分。虽然ADT余因数是 在自由状态下不稳定，正在开发以保护形式稳定它的方法。会是 同位素标记并结合到载脂蛋白酶中。这些实验将使我们能够识别 ADT的前体(主要假设：自由基SAM引起半胱氨酸-铁结合物的反应)。在.之前 Fe2(ADT)系综的构建，两个Fe-半胱氨酸-CO中心应该预先组织在一个 支架蛋白HydF。这项工作将产生第一个铁-半胱氨酸-CO络合物用于体外测试和 通过化学方法。最后，本工作将评估ADT在催化机理上的作用。的 特别感兴趣的是胺辅助因子的质子化状态对[2Fe]行为的影响 亚单位。后一项研究将在含有Fe2的模型络合物上进行， 修饰的胺辅基因子。特别感兴趣的是N质子化对氢原子和氢原子结合的影响 抑制剂CO和甲醛。 总的来说，这项工作的重点是了解最快的生物合成和作用机制 以从水中生产氢气而闻名的催化剂。

Hydrogenase enzymes are pervasive, being found in bacteria, archaea, and some higher organisms. Hydrogenases are found in many pathogens, including some that inhabit the human gut. The [FeFe] and [NiFe]-hydrogenases mediate the most fundamental chemical reaction: the interconversion of H2 with protons and reducing equivalents. Since H2 is an unusual substrate, the enzymes are also structurally exceptional with an array of distinctive cofactors, especially the site of H2 binding and release. Similarly, the biosynthesis of these active sites involves elaborate and novel biochemistry. In addition to biophysical approaches, elucidation of these mechanisms relies on organometallic chemistry, especially since the substrates (H2, H+, H-) are often invisible to conventional biophysical methods. This project aims to elucidate the biosynthesis and mechanism of action of the [FeFe] enzymes, the faster of the two main hydrogenases and the one most amenable to development for other applications. The work involves synthesis of proposed intermediates, spectroscopic and electrochemical characterization, in vitro assays, and isotopic labeling. It also relies on extensive collaborations with groups that offer specialized skills in theory, synchrotron-based spectroscopy, spin resonance, and in vitro testing. This project specifically focuses on the recently confirmed azadithiolate (adt) cofactor, which is the enzyme's most remarkable component from a mechanistic perspective. Although the adt cofactor is unstable in the free state, methods are being developed to stabilize it in protected form. It will be isotopically labeled and incorporated into an apo enzyme. These experiments will allow us to identify the precursor to the adt (main hypothesis: radical SAM induced reactions of cysteine-Fe conjugates). Prior to construction of the Fe2(adt) ensemble, two Fe-cysteine-CO centers are supposed to be preorganized in a scaffold protein HydF. This work will produce the first Fe-cysteine-CO complexes for testing in vitro and by chemical methods. Finally, the work will evaluate the role of adt on the catalytic mechanism. Of specific interest is the influence of the protonation state of the amine cofactor on the behavior of the [2Fe] subunit. The latter studies will be conducted on model complexes containing Fe2 with authentic and modified amine cofactors. Of specific interest is the influence of N-protonation on the binding of H2 and the inhibitors CO and formaldehyde. Overall, the work focuses on understanding the biosynthesis and mechanism of action of the fastest catalysts known for producing H2 from water.