Extensive ligand rearrangements around the [2Fe-2S] cluster of Clostridium pasteurianum ferredoxin.
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.
Département de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
M P
MP
Golinelli
C
C
Chatelet
E C
EC
Duin
M K
MK
Johnson
J
J
Meyer
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D003013:Clostridium / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet
C081714:2Fe-2S ferredoxin; D001426:Bacterial Proteins; D005288:Ferredoxins; D008024:Ligands; D003545:Cysteine