Iron-sulfur cofactors involved in metal center assembly and catalysis of hydrogenase

在优先计划（PP） 1927的第二个资助期，我们的联合项目将首先集中在阐明耐o2，耐热的NAD+还原[NiFe]氢化酶的异常活性位点性质及其对该复合FeS簇酶催化性质的影响。分子和机制细节将以合作的方式进行研究，这将涉及电子顺磁共振（EPR），红外（IR）和共振拉曼（RR）光谱，以识别和表征体外和体内催化相关的氧化还原状态。对相关的f420还原[NiFe]-氢化酶进行比较研究。在这种情况下，我们将应用最近建立的实验装置，该装置允许在受控的气体气氛下对相同的蛋白质晶体进行（低温）IR和RR测量。结合x射线晶体学和理论方法，这些实验将提供单个催化中间体的详细结构和电子信息。这种跨学科的方法将扩展到分析[Fe]氢化酶和氮化酶。在我们联合项目的第二部分，我们寻求提高对[NiFe]氢化酶的NiFe(CN-)2（CO）辅因子的多步生物合成过程的理解。我们将研究HypX在氧条件下通过甲酰基辅酶a将甲酰基四氢叶酸转化为[NiFe]氢化酶的活性位点CO配体的反应机理。实验计划揭示含FeS簇的HypCD成熟复合物的组成，该复合物能够接收（hypx生成的）CO分子，最终形成Fe(CN-)2（CO）单元。基于最近开发的策略，我们现在能够在催化中心的不同成熟阶段纯化分离的[NiFe]-氢化酶的大亚基。这种独特的情况使我们能够揭示NiFe(CN-)2（CO）辅因子组装的事件顺序。振动光谱技术将用于研究FeS中心和其他金属在氢化酶、氮酶（类）酶和复合体i的成熟和催化中的作用和相互作用。通过与PP联盟成员和外部合作伙伴的密切合作，我们将利用我们的结果来获得基于FeS的成熟和催化的总体原理的详细图像。

In the second funding period of the Priority Program (PP) 1927, our joint project will focus, first, on the elucidation of the unusual active site properties of an O2-tolerant, thermostable NAD+-reducing [NiFe]-hydrogenase and their impact on the catalytic properties of this complex FeS cluster enzyme. The molecular and mechanistic details will be investigated in a collaborative approach, which will involve electron paramagnetic resonance (EPR), infrared (IR), and resonance Raman (RR) spectroscopy to identify and characterize catalytically relevant redox states both in vitro and in vivo. Comparative studies will be performed on a related F420-reducing [NiFe]-hydrogenase. In this context, we will apply a recently established experimental setup, which allows (cryogenic) IR and RR measurements on the same protein crystal under controlled gas atmospheres. In combination with X-ray crystallography and theoretical methods, these experiments will provide detailed structural and electronic information on individual catalytic intermediates. This interdisciplinary approach will be extended to analyze [Fe] hydrogenase and nitrogenase. In the second part of our joint project, we seek to improve the understanding of the multistep biosynthesis process of the NiFe(CN-)2(CO) cofactor of [NiFe] hydrogenase. We will investigate the reaction mechanism by which HypX converts formyl-tetrahydrofolate via formyl-CoA into the active site CO ligand of [NiFe] hydrogenases under oxic conditions. Experiments are planned to unveil the composition of the FeS cluster-containing HypCD maturation complex competent in receiving the (HypX-generated) CO molecule to eventually form the Fe(CN-)2(CO) unit. Based on a recently developed strategy, we are now able to purify isolated large subunits of [NiFe]-hydrogenases in different maturation stages of the catalytic center. This unique situation allows us to unravel the sequence of events of NiFe(CN-)2(CO) cofactor assembly. Vibrational spectroscopic techniques will be used to investigate the role and interplay of FeS centers and other metals in the maturation and catalysis of hydrogenase, nitrogenase(-like) enzymes and Complex I. In close collaboration with members of the PP consortium and external partners, we will exploit our results to gain a detailed picture of the overarching principles of FeS-based maturation and catalysis.