Novel bio-synthetic constructs for the analysis and manipulation of the hydrogen catalysis of [FeFe]-hydrogenases

[FeFe]-氢酶是高效的产氢生物催化剂，因此有望成为开发新的制氢合成催化剂的候选对象。不幸的是，活性中心(H-簇)及其蛋白质环境的基本功能仍然知之甚少。这尤其适用于催化机制中的质子耦合电子传输方面，以及这类高效酶的氧敏感性问题。这些理解上的差距将通过在成熟和成熟的多学科合作框架内补充使用分子生物学、合成化学、光谱学和理论来弥合。一方面，将在25个定点突变变体的基础上寻求调节性质子转移和催化质子转移之间的机制和空间差异。另一方面，我们将获得野生型和突变型的样本系列，用于光谱监测依赖O2的H-簇在时间分辨的“冷冻-猝灭”过程中的降解过程。使用18O2进行H-簇展示，使我们能够通过质谱学跟踪生成的氧化产物(例如CN18O、C18O2或H218O)。此外，我们将尝试通过选择性地增加其气体通道系统中的分子筛效应来提高其中一种被检测酶(CPI)的耐氧性，从而产生更大的可变池并对该效应进行采样。为了建立一个全面的总体图景，所有的子项目都应用了蛋白质生物化学、结晶学、酶动力学和光谱技术(ATR-FTIR、XAS/XES、NRVS、FTIR、EPR和Mössbauer)以及量子力学计算。除了对蛋白质衍生物的研究外，还将利用H-簇结构中的元素替换来阐明所有配体在反应周期中的单独功能贡献以及它们对两个亚簇[4Fe]H和[2Fe]H的反应性(H+，e-，H2，O2)的影响。二硫代配体桥头的氮与磷的交换可以例如保持H-簇的质子导电性，同时避免在O2接触下形成活性氧物种(ROS)。还将研究金属专一性和H-簇的电子密度分布对O2诱导的H-簇降解过程的影响，采用新的金属同系物[Fe2(ADT)(CO)4(CN)2]2-辅因子通过体外成熟过程适合[FeFe]-氢酶CPI和HydA1的结合位，这是一项成熟且强大的技术，正在进行中。

[FeFe]-hydrogenases are highly efficient H2-producing biocatalysts and therefore promising candidates for blueprints for the development of new synthetic catalysts for H2 production. Unfortunately, essential functional aspects of the active center (H-cluster) and its protein environment are still poorly understood. This applies in particular to the aspects of proton-coupled electron transport in the catalytic mechanisms and the problem of the oxygen sensitivity of this otherwise highly efficient enzyme class. These gaps in comprehension are to be closed here through the complementary use of molecular biology, synthesis chemistry, spectroscopy and theory within the framework of a well-established and proven multidisciplinary cooperation. On the one hand, the mechanistic and spatial differentiation between regulatory and catalytic proton transfer will be pursued on the basis of 25 site directed mutagenesis variants. On the other hand, we will obtain sample series of wild type and mutant variants for the spectroscopic monitoring of the process of O2-dependent H-cluster degradation in a time-resolved "freeze-quench" procedure. The use of 18O2 for H-cluster exposition allows us to track the resulting oxidation products (e.g. CN18O, C18O2 or H218O) via mass-spectrometry. In addition, we will try to increase the O2 resistance of one of the examined enzymes (CpI) by selectively increasing of the molecular sieve effect in its gas channel system, whereby larger variant pools are generated and sampled on this effect. In order to create a comprehensive overall picture, protein-biochemistry, crystallographic, enzyme kinetic and spectroscopic techniques (ATR-FTIR, XAS/XES, NRVS, FTIR, EPR and Mössbauer) as well as quantum mechanical computations are applied in all sub-projects. In addition to the investigations on protein derivatives, element substitutions in the H-cluster architecture will be employed to elucidate the individual functional contributions of all ligands for the reaction cycle and their influence on the reactivity (H+, e-, H2, O2) concerning both sub-clusters, [4Fe]H and [2Fe]H. An exchange of nitrogen in the bridgehead of the dithiolate-ligand against phosphorus could e.g. maintain the proton conductivity to the H-cluster, while avoiding the formation of reactive oxygen species (ROS) under O2 contact. The influence of metal specificity and electron density distribution of the H-cluster for the process of O2-induced H-cluster degradation is also to be investigated, employing novel metal homologs of the [Fe2(ADT)(CO)4(CN)2]2- cofactor to be fitted into the binding niche of [FeFe]-hydrogenases CPI and HydA1 by the in vitro maturation procedure which is a well-established and powerful technique in the ongoing collaboration of both applicants.