Recombinant two-iron rubredoxin of Pseudomonas oleovorans: overexpression, purification and characterization by optical, CD and 113Cd NMR spectroscopies

摘要: The gene (alk G) encoding the two-iron rubredoxin of Pseudomonas oleovorans was amplified from genomic DNA by PCR and subcloned into expression vector pKK223-3. directed high-level production in Escherichia coli. A simple three-step procedure used to purify recombinant 1Fe form. 1Fe-rubredoxin readily converted 2Fe, apoprotein cadmium forms after precipitation with trichloroacetic acid resolubilization presence or absence ferrous ammonium sulphate CdCl2 respectively. Recombinant 2Fe rubredoxins are redox-active able transfer electrons reduced spinach ferredoxin reductase cytochrome c. absorption spectrum dichroic features CD for cadmium-substituted protein similar those reported Desulfovibrio gigas [Henehan, Poutney, Zerbe Vasak (1993) Protein Sci. 2, 1756-1764]. Difference spectroscopy revealed four Gaussian-resolved maxima at 207, 228, 241 280 nm; nm band is attributable, Jorgensen's electronegativity theory, a CysS-CdII charge-transfer excitation. 113Cd NMR 113Cd-substituted contains two resonances chemical shifts located 732.3 730 p.p.m. broader linewidth high frequency shift resonance p. p.m. indicates that Cd2+ ion undergoing exchange and, consistent difference spectra, bound less tightly than ion, giving rise