Expression of Desulfovibrio gigas desulforedoxin in Escherichia coli. Purification and characterization of mixed metal isoforms.
The dsr gene from Desulfovibrio gigas encoding the nonheme iron protein desulforedoxin was cloned using the polymerase chain reaction, expressed in Escherichia coli, and purified to homogeneity. The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D. gigas. These include an alpha 2 tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-to-iron charge transfer bands. Low temperature electron paramagnetic resonance studies confirm the presence of a high-spin ferric ion with g values of 7.7, 5.7, 4.1, and 1.8. Interestingly, E. coli produced two forms of desulforedoxin containing iron. One form was identified as a dimer with the metal-binding sites of both subunits occupied by iron while the second form contained equivalent amounts of iron and zinc and represents a dimer with one subunit occupied by iron and the second with zinc.
Czaja
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C
Section of Hematology Research, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Litwiller
R
R
Tomlinson
A J
AJ
Naylor
S
S
Tavares
P
P
LeGall
J
J
Moura
J J
JJ
Moura
I
I
Rusnak
F
F
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000096:biosynthesis; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D003901:Desulfovibrio / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli; D005798:Genes, Bacterial; D007501:Iron / Q000032:analysis / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013053:Spectrophotometry
D000596:Amino Acids; D001426:Bacterial Proteins; D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C022220:desulforedoxin; D007501:Iron