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首页> 外文期刊>Biochemistry >Heterologous biosynthesis and characterization of the [2Fe-2S]-containing N-terminal domain of Clostridium pasteurianum hydrogenase
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Heterologous biosynthesis and characterization of the [2Fe-2S]-containing N-terminal domain of Clostridium pasteurianum hydrogenase

机译：巴氏梭菌氢化酶含[2Fe-2S] N端结构域的异源生物合成和表征

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The primary structure of Clostridium pasteurianum hydrogenase I appears to be composed of modules suggesting that the various iron-sulfur clusters present in this enzyme might be segregated in structurally distinct domains. On the basis of this observation, a gene fragment encoding the 76 N-terminal residues of this enzyme has been expressed in Escherichia coli. The polypeptide thus produced contains a [2Fe-2S](n+) cluster of which the oxidized level (n = 2) has been monitored by UV-visible absorption, circular dichroism, and resonance Raman spectroscopy. This cluster can be reduced by dithionite or electrochemically to the n = 1 level which has been investigated by EPR and by low-temperature magnetic circular dichroism. The redox potential of the +2 to +1 transition is -400 mV (vs the normal hydrogen electrode). The spectroscopic and redox results indicate a [2Fe-2S](2+/+) chromophore coordinated by four cysteine ligands in a protein fold similar to that found in plant- and mammalian-type ferredoxins. Among the five cysteines present in the N-terminal hydrogenase fragment, four (in positions 34, 46, 49, and 62) are conserved in other sequences and are therefore the most likely ligands of the [2Fe-2S] site. The fifth cysteine, in position 39, can be dismissed on the grounds that the Cys39Ala mutation does not alter any of the properties of the iron-sulfur cluster. The spectroscopic signatures of this chromophore are practically identical with some of those reported for full-size hydrogenase. This confirms that C. pasteurianum hydrogenase I contains a [2Fe-2S] cluster and indicates that the polypeptide fold around the metal site of the N-terminal fragment is very similar, if not identical, to that occurring in the full-size protein. The N-terminal sequence of this hydrogenase is homologous to sequences of a number of proteins or protein domains, including a subunit of NADH-ubiquinone oxidoreductase of respiratory chains. From that, it can be anticipated that the structural domain isolated and described here is a building block of electron transfer complexes involved in various bioenergetic processes. [References: 46]
机译：巴氏梭菌加氢酶I的一级结构似乎由模块组成，表明该酶中存在的各种铁硫簇可能在结构上不同的域中分离。基于该观察，已经在大肠杆菌中表达了编码该酶的76个N-末端残基的基因片段。由此产生的多肽含有[2Fe-2S]（n +）簇，其氧化水平（n ＝ 2）已通过UV-可见吸收，圆二色性和共振拉曼光谱法监测。可通过连二亚硫酸盐或电化学方法将该团簇还原为n = 1的水平，这已通过EPR和低温磁性圆二色性进行了研究。 +2到+1跃迁的氧化还原电势为-400 mV（相对于正常氢电极）。光谱和氧化还原结果表明，[4Fe-2S]（2 + / +）发色团由四个半胱氨酸配体配位，其蛋白质折叠倍数类似于在植物型和哺乳动物型铁氧还蛋白中发现的。在N末端氢化酶片段中存在的五个半胱氨酸中，四个（在34、46、49和62位）在其他序列中是保守的，因此是[2Fe-2S]位点最可能的配体。可以以Cys39Ala突变不会改变铁硫簇的任何性质为理由，将位于位置39的第五个半胱氨酸排除。该生色团的光谱特征实际上与报道的全尺寸氢化酶的光谱特征相同。这证实巴氏梭菌加氢酶I含有[2Fe-2S]簇，并且表明围绕N末端片段的金属位点折叠的多肽与全尺寸蛋白质中发生的折叠非常相似，即使不是相同的话。该氢化酶的N-末端序列与许多蛋白质或蛋白质结构域的序列同源，包括呼吸链的NADH-泛醌氧化还原酶的亚基。由此可以预见，此处分离和描述的结构域是参与各种生物能过程的电子转移复合物的基础。 [参考：46]

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