Cell-free H-cluster Synthesis and [FeFe] Hydrogenase Activation: All Five CO and CN- Ligands Derive from Tyrosine
Cell-free H-cluster Synthesis and [FeFe] Hydrogenase Activation: All Five CO and CN- Ligands Derive from Tyrosine
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DOI：
10.1371/journal.pone.0020346
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发表时间：
2011-05-31
期刊：
影响因子：
3.7
通讯作者：
Swartz, James R.
中科院分区：
文献类型：
--
作者：
Kuchenreuther, Jon M.;George, Simon J.;Swartz, James R.
[FeFe]氢酶是作为可持续燃料和化学原料生产氢气的很有前途的催化剂，它们也是仿生放氢化合物的范例。氢的形成是由H-簇催化的，这是一种独特的铁基辅助因子，需要三个一氧化碳(CO)和两个氰化物(CN-)配体以及一个二硫代桥。三种辅助蛋白(Hyde、HydF和HydG)可能负责组装和安装Hcluster，但它们的确切作用和生物合成途径尚未完全确定。在这份报告中，我们描述了有效的无细胞方法来研究H-簇的合成和[FeFe]氢酶的激活。结合同位素标记和FTIR光谱，我们得出结论，CO和CN-分别来自酪氨酸的羧基和氨基。这种具有重组途径的体外系统为研究生物合成机制提供了一种通用的方法，这项工作标志着朝着理解H-簇组装和氢酶成熟所需的蛋白质相互作用和复杂反应迈出了重要的一步。
[FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN-) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the Hcluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN- ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the proteinprotein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.