Cysteine ligand swapping on a deletable loop of the [2Fe-2S] ferredoxin from Clostridium pasteurianum.
The [2Fe-2S] ferredoxin from Clostridium pasteurianum is unique among ferredoxins, both by its sequence and by the distribution of its cysteine residues (in positions 11, 14, 24, 56, and 60). In previous investigations, a combination of site-directed mutagenesis and of spectroscopic techniques showed that cysteines 11, 56, and 60 are ligands of the [2Fe-2S] cluster in the wild type protein and that cysteine 14 is not, but the status of cysteine 24 remained unclear. New mutated forms of this ferredoxin have been obtained and characterized. The data show that cysteine 24 is a ligand of the cluster in the wild type protein. When cysteine 24 is mutated into alanine, it is replaced as a cluster ligand by cysteine 14. The fourth ligand of the cluster can also be a cysteine residue newly introduced in position 16 when both cysteines 14 and 24 are replaced by alanine. These results suggest that the region encompassing cysteines 14 and 24 is a solvent-exposed flexible loop, in agreement with structure predictions. A number of nondeleterious deletions of variable length (3-14 residues) have been performed in the region of residues 17-32. The deletions were found to modify only marginally the spectroscopic properties of the [2Fe-2S] cluster but resulted in variations of its redox potential over a range of nearly 100 mV. This is the first instance of ligand swapping in a [2Fe-2S] protein, and the first time in any ferredoxin that a large loop has been excised from the structure without preventing the assembly of the iron-sulfur chromophore. Some of the molecular variants described here also highlight the similarities between the C. pasteurianum [2Fe-2S] ferredoxin and the 25 kDa subunit of the proton-translocating NADH: ubiquinone oxidoreductase of Paracoccus denitrificans.
Golinelli
M P
MP
Départment de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
Akin
L A
LA
Crouse
B R
BR
Johnson
M K
MK
Meyer
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003013:Clostridium / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D005798:Genes, Bacterial; D007501:Iron / Q000737:chemistry*; D008024:Ligands; D008280:Magnetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000737:chemistry; D017384:Sequence Deletion; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013455:Sulfur / Q000737:chemistry*
C081714:2Fe-2S ferredoxin; D005288:Ferredoxins; D008024:Ligands; D013455:Sulfur; D007501:Iron; D003545:Cysteine