A new [2Fe-2S] ferredoxin from Rhodobacter capsulatus. Coexpression with a 2[4Fe-4S] ferredoxin in Escherichia coli

A 285-base pair open reading frame was found immediately upstream of the fdxN gene (encoding ferredoxin I) of Rhodobacter capsulatus and coded for a 95-amino acid protein with a predicted molecular weight of 10,156. The deduced amino acid sequence contained 5 cysteines, 4 of which exhibited spacing...

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Bibliographic Details
Published in:The Journal of biological chemistry 1991-02, Vol.266 (5), p.3294-3299
Main Authors: Grabau, C, Schatt, E, Jouanneau, Y, Vignais, P M
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acids - analysis
Bacteria - genetics
Bacteria - metabolism
Blotting, Northern
Chromatography, DEAE-Cellulose
DNA, Bacterial - genetics
Electron Spin Resonance Spectroscopy
Escherichia coli - genetics
ferredoxin
Ferredoxins - biosynthesis
Gene Expression Regulation, Bacterial
genes
Genes, Bacterial
Molecular Sequence Data
Plants - genetics
Restriction Mapping
Sequence Homology, Nucleic Acid
Spectrophotometry, Ultraviolet
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Summary:A 285-base pair open reading frame was found immediately upstream of the fdxN gene (encoding ferredoxin I) of Rhodobacter capsulatus and coded for a 95-amino acid protein with a predicted molecular weight of 10,156. The deduced amino acid sequence contained 5 cysteines, 4 of which exhibited spacing characteristic of [2Fe-2S] plant and cyanobacterial ferredoxins. The amino acid sequence was found to share approximately 25% amino acid similarity with plant-type ferredoxins. The gene was named fdxC. Expression of the fdxC and fdxN genes together in Escherichia coli was accomplished by subcloning the genes in the vector pUC18 downstream of the lac promoter. Cells containing this plasmid produced a red and a brown protein corresponding to the fdxC and fdxN gene products, respectively. EPR and UV-visible absorption spectroscopy confirmed that the FdxC protein contained a [2Fe-2S] cluster and the FdxN protein contained two [4Fe-4S] clusters and that the centers were correctly assembled and inserted in the ferredoxins expressed in E. coli. Transcription (Northern blot) analysis showed that the genes were transcribed only under nitrogen-limiting (nif-derepressing) growth conditions.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)49987-5