Recombinant two-iron rubredoxin of Pseudomonas oleovorans: overexpression, purification and characterization by optical, CD and 113Cd NMR spectroscopies.
The gene (alk G) encoding the two-iron rubredoxin of Pseudomonas oleovorans was amplified from genomic DNA by PCR and subcloned into the expression vector pKK223-3. The vector directed the high-level production of rubredoxin in Escherichia coli. A simple three-step procedure was used to purify recombinant rubredoxin in the 1Fe form. 1Fe-rubredoxin was readily converted to the 2Fe, apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of ferrous ammonium sulphate or CdCl2 respectively. Recombinant 1Fe and 2Fe rubredoxins are redox-active and able to transfer electrons from reduced spinach ferredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the cadmium-substituted protein are similar to those reported for cadmium-substituted Desulfovibrio gigas rubredoxin [Henehan, Poutney, Zerbe and Vasak (1993) Protein Sci. 2, 1756-1764]. Difference absorption spectroscopy of cadmium-substituted rubredoxin revealed the presence of four Gaussian-resolved maxima at 207, 228, 241 and 280 nm; the 241 nm band is attributable, from Jorgensen's electronegativity theory, to a CysS-CdII charge-transfer excitation. The 113Cd NMR spectrum of the 113Cd-substituted rubredoxin contains two 113Cd resonances with chemical shifts located at 732.3 and 730 p.p.m. The broader linewidth and high frequency shift of the resonance at 730 p. p.m. indicates that the Cd2+ ion is undergoing chemical exchange and, consistent with the difference absorption spectra, is bound less tightly than the Cd2+ ion, giving rise to the chemical shift at 732.3 p.p.m.
Lee
H J
HJ
Department of Biochemistry, University of Leicester, U.K.
Lian
L Y
LY
Scrutton
N S
NS
D002104:Cadmium / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D007554:Isotopes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010084:Oxidation-Reduction; D011199:Potentiometry; D011549:Pseudomonas / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D012416:Rubredoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D007554:Isotopes; D011994:Recombinant Proteins; D012416:Rubredoxins; D002104:Cadmium