H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG
H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG
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DOI：
10.1007/s00775-019-01709-7
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发表时间：
2019-09-01
影响因子：
3
通讯作者：
Broderick, Joan B.
中科院分区：
文献类型：
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作者：
Byer, Amanda S.;Shepard, Eric M.;Broderick, Joan B.
[FeFe]-氢酶催化质子在一个复杂的金属辅助因子位置H-簇上可逆还原为H-2。这个活性中心H-簇的生物合成需要三个成熟酶：自由基腺苷蛋氨酸酶Hyde和HydG合成非蛋白质配体，而GTPase HydF为H-簇的2FE亚簇([2Fe](H))的组装提供支架，然后将其转移到氢酶。为了描述与HydF([2Fe](F))配位的2FE前体簇的组装和传递步骤，我们在单独HydF(E)或单独HydG(G)的情况下异源表达了HydF，并使用UV-可见、EPR和FTIR光谱和生化分析对得到的纯化的HydF(E)和HydF(G)进行了表征。通过与Hyde或HydG共表达来修饰HydF上的铁硫簇合物，可见、EPR和FTIR光谱特征的变化证明了这一点。此外，生化分析表明，HydF(E)能够在一定程度上激活HydA(Delta EFG)(类似于WT的1%)，即使没有[2Fe](H)(HydG)的正常CO和CN-配体来源。相比之下，用HydF(G)进行的激活分析显示没有成熟HydA(Delta EFG)的能力。在HydF(E)的情况下，在没有HydG的情况下，来自细胞环境的微量硅藻类化合物被结合到HydF上的类似[2Fe](F)的前体中。我们的结论是，Hyde的产物，可能是[2Fe](H)的二硫甲胺配体，对于活化过程是绝对必要的，而HydG的双原子产物可以从其他来源提供。图形摘要
[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H-2 at a complex metallocofactor site, the H-cluster. Biosynthesis of this active-site H-cluster requires three maturation enzymes: the radical S-adenosylmethionine enzymes HydE and HydG synthesize the nonprotein ligands, while the GTPase HydF provides a scaffold for assembly of the 2Fe subcluster of the H-cluster ([2Fe](H)) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe](F)), we have heterologously expressed HydF in the presence of HydE alone (HydF(E)) or HydG alone (HydF(G)), and characterized the resulting purified HydF(E) and HydF(G) using UV-visible, EPR, and FTIR spectroscopies and biochemical assays. The iron-sulfur clusters on HydF are modified by co-expression with HydE or HydG, as evidenced by the changes in the visible, EPR, and FTIR spectral features. Further, biochemical assays show that HydF(E) is capable of activating HydA(Delta EFG) to a limited extent (similar to 1% of WT) even though the normal source of CO and CN- ligands of [2Fe](H) (HydG) was absent. Activation assays performed with HydF(G), in contrast, exhibit no ability to mature HydA(Delta EFG). It appears that in the case of HydF(E), trace diatomics from the cellular environment are incorporated into a [2Fe](F)-like precursor on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe](H), is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources. Graphic abstract