Abstract
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.
Highlights
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 presence of four cysteine residues! subunit in desulforedoxin has led to a model in which the iron is coordinated by four cysteinyl sulfur atoms, analogous to the tetrahedrally coordinated iron atom of rubredoxin [6]
We describe the cloning of the dsr' gene from D. gigas and the expression of the recombinant protein in Escherichia coli
Summary
The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D. gigas These include an lr tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-toiron charge transfer bands. The crystal structure of desulforedoxin, recently determined to 1.8-A resolution, has found that the desulforedoxin metal is coordinated by four cysteine residues in a tetrahedral arrangement significantly distorted when compared with rubredoxin [13]. At least two distinct species were resolved during purification One of these was identified as a dimer with the metal site of each subunit occupied by iron and is presumably identical to the D. gigas protein previously isolated [1,2,3,4]. This species represents a mixed-metal iron/zinc dimer in which zinc has been incorporated into one of the iron-binding sites during expression
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