Golinelli M P, Chatelet C, Duin E C, Johnson M K, Meyer J
Département de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
Biochemistry. 1998 Jul 21;37(29):10429-37. doi: 10.1021/bi9806394.
The [2Fe-2S] cluster of the ferredoxin from Clostridium pasteurianum is coordinated by cysteines 11, 56, and 60 and by a fourth cysteine, residue 24 in the wild-type protein, located on a flexible and deletable loop around residues 14-30. New mutated forms of this ferredoxin show that the fourth cysteine ligand can be located in any one of positions 14, 16, 21, 24, or 26. Another set of molecular variants has unveiled a new case of ligand swapping on the cysteine 60 ligand site. Replacement of cysteine 60 by alanine and introduction of a cysteine in position 21 yielded a ferredoxin that assembles a [2Fe-2S] cluster of which the ligands are cysteines 11, 21, 24, and 56. This cysteine ligand pattern is similar to that occurring in plant-type or mammalian-type ferredoxins, although the overall sequence similarities are below detection. Moreover, the vibrational and electronic properties of the resulting [2Fe-2S]2+/+ center, as revealed by resonance Raman and EPR studies, are strikingly similar to those of mammalian-type ferredoxins. The extensive set of mutated forms of the C. pasteurianum ferredoxin now available indicates that cysteine ligand exchange may occur on residues 24 and 60, but not on residues 11 and 56. It is thus suggested that cysteines 24 and 60 are part of a solvent accessible aspect of the Fe-S cluster, whereas cysteines 11 and 56 are buried and form the more rigid part of the polypeptide ligand framework. In view of the unprecedented versatility of this [2Fe-2S] cluster and of its polypeptidic environment, the introduction of ligands other than cysteine in various positions has been attempted. These experiments have remained unsuccessful, and even including previous studies, noncysteinyl ligation has been obtained with this protein in only very few cases. The data provide an extensive confirmation that Fe-S clusters have a strong preference for thiolate ligation and rationalize the relatively rare occurrence of noncysteinyl ligation in native Fe-S proteins.
巴斯德梭菌铁氧化还原蛋白的[2Fe-2S]簇由半胱氨酸11、56和60以及第四个半胱氨酸（野生型蛋白中的第24位残基）配位，该第四个半胱氨酸位于14 - 30位残基周围的一个灵活且可缺失的环上。这种铁氧化还原蛋白的新突变形式表明，第四个半胱氨酸配体可以位于第14、16、21、24或26位中的任何一个位置。另一组分子变体揭示了半胱氨酸60配体位点上配体交换的一个新例子。将半胱氨酸60替换为丙氨酸并在第21位引入一个半胱氨酸，得到了一种铁氧化还原蛋白，它组装了一个[2Fe-2S]簇, 其配体为半胱氨酸11、21、24和56。这种半胱氨酸配体模式类似于植物型或哺乳动物型铁氧化还原蛋白中的模式，尽管总体序列相似性低于检测水平。此外，通过共振拉曼和电子顺磁共振研究揭示，所得[2Fe-2S]2+/+中心的振动和电子性质与哺乳动物型铁氧化还原蛋白的惊人相似。目前可得的大量巴斯德梭菌铁氧化还原蛋白的突变形式表明，半胱氨酸配体交换可能发生在第24和60位残基上，但不会发生在第11和56位残基上。因此，有人提出半胱氨酸24和60是Fe-S簇溶剂可及部分的一部分，而半胱氨酸11和56则被掩埋并形成多肽配体框架中更刚性的部分。鉴于这种[2Fe-2S]簇及其多肽环境前所未有的多功能性，人们尝试在不同位置引入除半胱氨酸以外的配体。这些实验均未成功，即使包括之前的研究，用这种蛋白仅在极少数情况下获得了非半胱氨酸连接。这些数据广泛证实了Fe-S簇对硫醇盐连接有强烈偏好，并解释了天然Fe-S蛋白中非半胱氨酸连接相对罕见的现象。
