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

氢化酶是普遍存在的，存在于细菌、古细菌和一些高等生物中。 氢化酶存在于许多病原体中，包括一些栖息在人类肠道中的病原体。[001 pdf 1st-31files] [NiFe]-氢化酶介导最基本的化学反应：H2与 质子和还原当量。由于H2是一种不寻常的底物，因此这些酶在结构上也是 一系列独特的辅因子，特别是H2结合和释放的位点。同样地， 这些活性位点的生物合成涉及复杂和新颖的生物化学。除了 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-）通常对于常规生物物理方法是不可见的。 该项目旨在阐明[FeFe]酶的生物合成和作用机制， 这是两种主要氢化酶中最适合开发用于其他应用的一种。的 工作涉及所提出的中间体的合成，光谱和电化学表征， 体外分析和同位素标记。它还依赖于与团体的广泛合作， 在理论、同步加速器光谱学、自旋共振和体外测试方面的专业技能。 该项目特别关注最近证实的azadithiolate（adt）辅因子， 从机械学的角度来看酶最显著的成分。虽然adt辅因子是 在自由状态下不稳定，正在开发方法以使其以受保护的形式稳定。将 同位素标记并掺入到载脂蛋白酶中。这些实验将使我们能够识别 adt的前体（主要假设：自由基SAM诱导的半胱氨酸-Fe缀合物反应）。之前 在Fe 2（adt）系综的构建中，两个Fe-半胱氨酸-CO中心被认为是预组织在一个 支架蛋白HydF。这项工作将产生第一个用于体外测试的Fe-半胱氨酸-CO复合物， 通过化学方法。最后，本工作将评估adt在催化机制上的作用。的 特别感兴趣的是胺辅因子的质子化状态对[2Fe] 亚单位后面的研究将在含有Fe 2的模型络合物上进行， 修饰的胺辅因子。特别令人感兴趣的是N-质子化对H2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生物合成和最快的行动机制， 已知用于由水产生H2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.