Biochemical and spectroscopic characterization of overexpressed fuscoredoxin from Escherichia coli.
Fuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfido-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mössbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data.
Pereira
A S
AS
Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Monte de Caparica, 2825-114, Portugal.
Tavares
P
P
Krebs
C
C
Huynh
B H
BH
Rusnak
F
F
Moura
I
I
Moura
J J
JJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D004926:Escherichia coli / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015204:Spectroscopy, Mossbauer
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C111122:fuscoredoxin protein, Desulfovibrio desulfuricans