The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]-hydrogenases
The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]-hydrogenases
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DOI：
10.1016/j.jmb.2004.09.040
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发表时间：
2004-11-12
影响因子：
5.6
通讯作者：
Böck, A
中科院分区：
文献类型：
--
作者：
Blokesch, M;Albracht, SPJ;Böck, A
氨甲酰磷酸已被证明是用于合成来自大肠杆菌的氢化酶的NiFe金属中心的CN配体的离析物。在缺乏氨甲酰磷酸的情况下，细胞积累两种氢化酶成熟蛋白的复合物，即用于合成氢化酶3的HypC和HypD。已经开发了纯化野生型HypD蛋白或在N末端携带Strep-tagII（（R））的生物活性衍生物的方法。HypD是一种单体蛋白质，每摩尔蛋白质含有约4摩尔铁。电子顺磁共振（EPR）和穆斯堡尔谱表明，铁以反磁性[4Fe-4S]（2+）团簇形式存在。HypC和HypD之间的复合物可以通过许多硫醇和伯胺特异性接头交联。当HypD和HypC与HypE并排过量产生时，HypC-HypD复合物含有亚化学计量量的HypE，当HypF也过量产生时，其在复合物中的比例可以增加。捕获在该复合物中的HypE可以被蛋白质HypF氨甲酰化，脱水后将氰基转移到复合物的HypC-HypD部分。游离HypC和HypD不被HypE-CN氰化。来自厌氧细胞的活性HypC-HypD复合物通过与K-3[Fe（CN）（6）]孵育而失活，但不与K-4[Fe（CN）（6）]孵育。结果表明，氢化酶成熟蛋白HypD，HypC，HypE和HypF，这是配体生物合成和连接到氢化酶3中的NiFe位点的铁原子的位点之间的动态复合物的存在。(C)2004爱思唯尔有限公司保留所有权利。
Carbamoylphosphate has been shown to be the educt for the synthesis of the CN ligands of the NiFe metal centre of hydrogenases from Escherichia coli. In the absence of carbamoylphosphate, cells accumulate a complex of two hydrogenase maturation proteins, namely HypC and HypD for the synthesis of hydrogenase 3. A procedure for the purification of wild-type HypD protein or of a biologically active derivative carrying the Strep-tagII((R)) at the N terminus has been developed. HypD is a monomeric protein possessing about 4 mol of iron per mol of protein. Electron paramagnetic resonance (EPR) and Mossbauer spectroscopy demonstrated that the iron is present as a diamagnetic [4Fe-4S](2+) cluster. The complex between HypC and HypD can be cross-linked by a number of thiol and primary amine-specific linkers. When HypD and HypC were overproduced side-by-side with HypE, the HypC-HypD complex contained substoichiometric amounts of HypE whose proportion in the complex could be augmented when HypF was also overproduced. HypE trapped in this complex could be carbamoylated by protein HypF and after dehydration transferred the cyano group to the HypC-HypD part of the complex. Free HypC and HypD were not cyanated by HypE-CN. An active HypC-HypD complex from anaerobic cells was inactivated by incubation with K-3[Fe(CN)(6)] but not with K-4[Fe(CN)(6)]. The results suggest the existence of a dynamic complex between the hydrogenase maturation proteins HypD, HypC, HypE and HypF, which is the site of ligand biosynthesis and attachment to the iron atom of the NiFe site in hydrogenase 3. (C) 2004 Elsevier Ltd. All rights reserved.