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

[NiFe]-氢化酶是一种铁硫（FeS）酶，在大亚基的活性位点有一个双金属的NiFe(CN)2CO辅因子，由四个半胱氨酸硫醇协调。辅因子由6个保守的Hyp蛋白合成。辅助因子Fe(CN)2CO部分的合成在HypCDEF蛋白支架上完成，然后插入到大亚基的前体中。HypD形成支架复合物的核心，并通过两个保守的半胱氨酸巯基与HypC一起配位Fe(CN)2CO基团；一个来自HypD (C41)，一个来自HypC （C2）。氰化物配体是由氨基甲酰转移酶HypF和脱水酶HypE共同作用产生的。CO配体与辅因子上的Fe的合成路线仍未确定，当它被添加时也是如此，但我们的工作假设是，这源于先前显示的与Fe协调的CO2。HypD有一个[4Fe-4S]簇，在上一个资助期内，我们确定了簇的氧化还原电位为Eo ' = -260 mV。我们还证明了两个保守的Cys残基（C69和C72）在两个连续的单电子和质子转移步骤中进行了二硫化物-硫醇交换。此外，我们发现HypD具有atp酶活性（ADP形成），这取决于Cys41。在试图确定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还蛋白中的一种是否起这种作用。利用蛋白-蛋白相互作用研究结合天然质谱法，我们发现只有HypC的一种旁联物HybG与大亚基前体相互作用。HypD不与HybG和大亚基前体进入三元络合物。此外，HybG只与前体相互作用，而不与c末端加工的成熟大亚基相互作用。因此，HypC/HybG进入与HypD或大亚基前体的复合物中，并在Fe(CN)2CO基团的构建和通过硫醇转移酶反应将其最终递送到靶蛋白中发挥作用。在下一个资助期，我们将更详细地描述HypD的atp酶活性。我们将确定哪些氨基酸和基序对HypD的atp酶活性是必需的。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，即该酶可以减少二氧化碳（Eo ' = ca.-530 mV）或可能是羧酸基，并且使用我们开发的ATP酶试验，我们将尝试证明这种活性。进一步的实验将集中在双原子配体的加成顺序，HypC将Fe-(CN)2CO基团转移到大亚基上，以及确定HypD和HypC的进一步相互作用伙伴，以深入了解Fe离子的体内供体以及CO配体的前体代谢产物。

[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.