Towards a mechanistic understanding of the role of the iron-sulphur cluster-containing HypD protein in diatomic ligand biosynthesis of [NiFe]-hydrogenases

深入了解含铁硫簇的 HypD 蛋白在 [NiFe]-氢化酶双原子配体生物合成中的作用

• 批准号: 310984010
• 负责人: Professor Dr. Gary Sawers
• 金额: --
• 依托单位: Institutsbereich Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/310984010?language=en
• 关键词: Towards; mechanistic; understanding; role; iron

[NiFe]-hydrogenases are iron-sulphur (FeS) enzymes that have a bimetallic NiFe(CN)2CO cofactor coordinated by four cysteinyl thiols in the active site of the large subunit. The cofactor is synthesized by six conserved Hyp proteins. Synthesis of the Fe(CN)2CO portion of the cofactor is completed on a HypCDEF protein scaffold and is then inserted into a precursor of the large subunit. HypD forms the core of the scaffold complex and together with HypC coordinates the Fe(CN)2CO group via two conserved cysteinyl thiols; one from HypD (C41) and one from HypC (C2). The cyanide ligands are generated by the combined actions of the carbamoyltransferase HypF and the dehydratase HypE. The synthesis route of the CO ligand to the Fe on the cofactor is still unresolved, as is when it is added, but our working hypothesis is that this originates from CO2 previously shown to be coordinated to the Fe. HypD has a [4Fe-4S] cluster and in the last funding period we have determined the redox potential of the cluster to be Eo’ = -260 mV. We have also shown that two conserved Cys residues (C69 and C72) undergo disulfide-thiol exchange in two consecutive one-electron and -proton transfer steps. Moreover, we showed that HypD has an ATPase activity (ADP is formed), which depends on Cys41. While trying to identify the electron donor to HypD’s FeS cluster, we could exclude ferredoxin in this capacity and we are currently analyzing whether one of the two flavodoxins found in Escherichia coli functions in this role. Using protein-protein interaction studies combined with native mass spectrometry, we showed that only HybG, a paralogue of HypC, interacts with the large subunit precursor. HypD does not enter into a ternary complex with HybG and the large subunit precursor. Moreover, HybG only interacts with the precursor and not the C-terminally processed, mature large subunit. Thus, HypC/HybG enters into a complex either with HypD or with the large subunit precursor and functions both in the construction of the Fe(CN)2CO group and its final delivery to the target protein via a thiol transferase reaction. In the next funding period, we will characterize the ATPase activity of HypD in more detail. We will determine which amino acids and motifs are required for the ATPase activity of HypD. We will also determine whether the interaction of HypD with HypC/HybG influences ATP hydrolysis. The fact that ATP is hydrolyzed by HypD is congruent with our working hypothesis that the enzyme reduces CO2 (Eo’ = ca.-530 mV) or possibly a carboxylate group and using the ATPase assay we have developed, we will attempt to demonstrate this activity. Further experiments will focus on the order-of-addition of the diatomic ligands, the transfer of the Fe-(CN)2CO group by HypC to the large subunit and the identification of further interaction partners of both HypD and HypC, to provide insight into the in vivo donors of both the Fe ion as well as the precursor metabolite of the CO ligand.

[NiFe]-氢化酶是铁硫（FeS）酶，具有双金属NiFe（CN）2CO辅因子，由大亚基活性位点中的四个半胱氨酰硫醇配位。辅因子由六个保守的Hyp蛋白合成。辅因子的Fe（CN）2CO部分的合成在HypCDEF蛋白支架上完成，然后插入大亚基的前体中。HypD形成支架复合物的核心，并与HypC一起通过两个保守的半胱氨酰硫醇配位Fe（CN）2CO基团;一个来自HypD（C41），一个来自HypC（C2）。氰化物配体通过氨甲酰转移酶HypF和乙酰化酶HypE的联合作用产生。CO配体与辅因子上的Fe的合成路线仍然没有得到解决，因为当它被添加时，但我们的工作假设是，这源于先前显示的与Fe配位的CO2。HypD具有[4Fe-4S]簇，并且在上一个资助期中，我们已经确定簇的氧化还原电位为E0 ' = -260 mV。我们还表明，两个保守的半胱氨酸残基（C69和C72）进行二硫键巯基交换在两个连续的单电子和质子转移步骤。此外，我们发现HypD具有ATP酶活性（形成ADP），这取决于Cys 41。在试图识别HypD的FeS簇的电子供体时，我们可以排除铁氧还蛋白的这种能力，我们目前正在分析大肠杆菌中发现的两种黄氧素之一是否具有这种作用。使用蛋白质-蛋白质相互作用的研究结合天然质谱，我们发现，只有HybG，一个parastrant的HypC，与大亚基前体相互作用。HypD不与HybG和大亚基前体形成三元复合物。此外，HybG仅与前体相互作用，而不与C末端加工的成熟大亚基相互作用。因此，HypC/HybG与HypD或大亚基前体形成复合物，并在Fe（CN）2CO基团的构建及其通过巯基转移酶反应最终递送至靶蛋白中发挥作用。在下一个资助期，我们将更详细地描述HypD的ATP酶活性。我们将确定HypD的ATP酶活性所需的氨基酸和基序。我们还将确定HypD与HypC/HybG的相互作用是否影响ATP水解。ATP被HypD水解的事实与我们的工作假设是一致的，即酶减少CO2（Eo' = ca. 530 mV）或可能是羧酸基团，并使用我们开发的ATP酶测定法，我们将尝试证明这种活性。进一步的实验将集中在订单的添加的双原子配体，转移的Fe-（CN）2CO组由HypC大亚基和鉴定进一步的相互作用的合作伙伴的HypD和HypC，提供洞察到体内捐助者的Fe离子以及前体代谢产物的CO配体。