Atta M, Lafferty M E, Johnson M K, Gaillard J, Meyer J
Département de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
Biochemistry. 1998 Nov 10;37(45):15974-80. doi: 10.1021/bi9812928.
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.
巴氏梭菌氢化酶I的一级结构似乎由多个模块组成，这表明该酶中存在的各种铁硫簇可能被分隔在结构不同的结构域中。基于这一观察结果，编码该酶76个N端残基的基因片段已在大肠杆菌中表达。由此产生的多肽含有一个[2Fe-2S]n+簇，其氧化态（n = 2）已通过紫外可见吸收、圆二色性和共振拉曼光谱进行监测。该簇可以通过连二亚硫酸盐或电化学方法还原为n = 1态，已通过电子顺磁共振和低温磁圆二色性对其进行了研究。+2到+1转变的氧化还原电位为-400 mV（相对于标准氢电极）。光谱和氧化还原结果表明，一个[2Fe-2S]2+/+发色团由四个半胱氨酸配体配位，其蛋白质折叠结构类似于植物型和哺乳动物型铁氧还蛋白中的结构。在N端氢化酶片段中存在的五个半胱氨酸中，有四个（位于第34、46、49和62位）在其他序列中是保守的，因此最有可能是[2Fe-2S]位点的配体。第39位的第五个半胱氨酸可以排除，因为Cys39Ala突变不会改变铁硫簇的任何性质。该发色团的光谱特征与报道的全尺寸氢化酶的一些特征几乎相同。这证实了巴氏梭菌氢化酶I含有一个[2Fe-2S]簇，并表明N端片段金属位点周围的多肽折叠结构即使不完全相同，也与全尺寸蛋白质中的结构非常相似。该氢化酶的N端序列与许多蛋白质或蛋白质结构域的序列同源，包括呼吸链中NADH-泛醌氧化还原酶的一个亚基。由此可以推测，这里分离和描述的结构域是参与各种生物能量过程的电子传递复合物的一个构建模块。
