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

[FeFe]-氢化酶是高效的H2生产生物催化剂，因此有前途的候选人的蓝图，用于开发新的合成催化剂的H2生产。不幸的是，活性中心（H簇）及其蛋白质环境的基本功能方面仍然知之甚少。这特别适用于催化机制中的质子偶联电子传递方面，以及这种高效酶类的氧敏感性问题。这些理解上的差距将在这里通过互补使用分子生物学，合成化学，光谱学和理论在一个完善的和经过验证的多学科合作的框架内被关闭。一方面，将在25个定点突变变体的基础上追求调节和催化质子转移之间的机制和空间差异。另一方面，我们将获得一系列的野生型和突变体的光谱监测过程中的O2依赖的H-簇降解的时间分辨的“冷冻淬火”程序的样品。使用18 O2进行H-团簇暴露使我们能够通过质谱法追踪所得的氧化产物（例如CN 18 O、C18 O2或H218 O）。 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增加所检查的酶之一（CpI）的耐氧性，从而产生更大的变体池并在该效应上取样。为了创造一个全面的整体画面，蛋白质生物化学，晶体学，酶动力学和光谱技术（ATR-FTIR，XAS/XES，NRVS，FTIR，EPR和穆斯堡尔）以及量子力学计算应用于所有子项目。除了对蛋白质衍生物的研究外，H-簇结构中的元素取代将被用来阐明所有配体对反应循环的单独功能贡献以及它们对关于两个亚簇[4Fe]H和[2Fe]H的反应性（H+，e-，H2，O2）的影响。二硫代硫酸盐-配体的桥头中的氮与磷的交换可以例如保持到H-簇的质子传导性，同时避免在O2接触下形成活性氧物质（ROS）。 还研究了金属特异性和H-团簇的电子密度分布对O2诱导H-团簇降解过程的影响，使用[Fe 2（ADT）（CO）4（CN）2]2-辅因子的新金属同系物，通过体外成熟程序将其装配到[FeFe]-氢化酶CPI和HydA 1的结合生态位中，在双方申请人的持续合作中建立了强大的技术。

[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.