Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies.
A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively. In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci. 2, 1756-1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276 nm; from Jørgensen's electronegativity theory, the 240 nm band is attributable to a CysS-Cd(II) charge-transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However, the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin.
Perry
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Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LE1 7RH, U.K.
Lian
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Scrutton
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry; D002942:Circular Dichroism; D017931:DNA Primers; D006868:Hydrolysis; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000378:metabolism*; D012416:Rubredoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D012416:Rubredoxins; D002104:Cadmium; D007501:Iron