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 Pseudomo nas oleovorans was created from the parental alk G gene contained in the ex pression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin: a simple procedure was used to p urify the recombinant domain in the 1Fe form. The 1Fe form of the C-termina l domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presenc e or absence of cadmium chloride respectively, In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer elec trons from reduced rubredoxin reductase to cytochrome c, The absorption spe ctrum and dichroic features of the CD spectrum for the iron- and cadmium-su bstituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zer be and Vasak (1993) Protein Sci, 2, 1756-1764]. Difference absorption spect roscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276 nm; from Jorgense n's electronegativity theory, the 240 nm band is attributable to a CysS-Cd( II) charge transfer excitation. Attempts to express the N-terminal domain o f the 2Fe rubredoxin directly from a minigene were unsuccessful. However: t he N-terminal domain was isolated through cleavage of an engineered 2Fe rub redoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by abso rption spectra typical of the 1Fe rubredoxins. The domain is folded as dete rmined by CD and NMR spectroscopies and is redox-active. However, the N-ter minal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-sub stituted Ps. oleovorans rubredoxin.