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

用于分析和操作[FeFe]-氢化酶氢催化的新型生物合成结构

• 批准号: 436793189
• 负责人: Professor Dr. Ulf-Peter Apfel
• 金额: --
• 依托单位: Anorganische Chemie I: Lehrstuhl für Bioanorganische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/436793189?language=en
• 关键词: Novel; bio; synthetic; constructs; analysis

[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.

[FeFe]-氢化酶是高效的产氢生物催化剂，因此有望成为开发新型产氢合成催化剂的蓝图候选者。不幸的是，人们对活性中心（H 簇）的基本功能及其蛋白质环境仍然知之甚少。这尤其适用于催化机制中质子耦合电子传输的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通过在完善和经过验证的多学科合作框架内互补使用分子生物学、合成化学、光谱学和理论来弥补。一方面，将在 25 个定点诱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，我们将获得野生型和突变体的样品系列，用于在时间分辨的“冷冻-猝灭”程序中对 O2 依赖性 H 团簇降解过程进行光谱监测。使用 18O2 进行 H 团簇暴露使我们能够通过质谱追踪所得的氧化产物（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增加所检查的酶（CpI）之一的O2抵抗力，从而根据该效应生成和采样更大的变体库。为了创建全面的整体图景，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之外，H簇结构中的元素替换还将用于阐明所有配体对反应循环的各自功能贡献及其对两个子簇[4Fe]H和[2Fe]H反应性（H+、e-、H2、O2）的影响。例如，二硫醇盐-配体桥头中的氮与磷的交换可以保持质子对 H 团簇的传导性，同时避免在 O2 接触下形成活性氧 (ROS)。 H簇的金属特异性和电子密度分布对O2诱导的H簇降解过程的影响也有待研究，采用[Fe2(ADT)(CO)4(CN)2]2-辅因子的新型金属同系物通过体外成熟程序安装到[FeFe]-氢化酶CPI和HydA1的结合位中，这是一个 在双方申请人的持续合作中建立了完善且强大的技术。