Specific Overproduction and Purification of the Cytochrome b₅₅₈ Component of the Cytochrome d Complex from Escherichia coli

In Escherichia coli strain GR84N[pNG10], the cloned gene for subunit I of the membrane-bound cytochrome d complex resulted in the overproduction of cytochrome b₅₅₈ and facilitated purification of this cytochrome. Extracting membranes with 1% Triton X-100 followed by two chromatographic steps yielded a single band on sodium dodecyl sulfate-polyacrylamide gels corresponding to subunit I (M_r57000). Purified cytochrome b₅₅₈ was in its native state as determined by difference absorption spectroscopy and by potentiometric analysis. Both the membranes of strain GR84N[pNG10] and the purified subunit I lacked the other two spectroscopically defined cytochromes, b₅₉₅(previously “a₁”) and d, of the cytochrome d complex. Reconstitution of cytochrome b₅₅₈ in phospholipid vesicles demonstrated that cytochrome b₅₅₈ can be reduced by ubiquinol but that it does not reduce molecular oxygen. Heme extraction of cytochrome b₅₅₈ yielded an extinction coefficient of 22000 M^-1 cm^-1 for the wavelength pair of 560 and 580 nm in the reduced-minus-oxidized spectrum. The mutation on pNG10 that eliminates subunit II was mapped to a 250 base pair DNA fragment.