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

George N. Green, Robert M. Lorence, Robert B. Gennis

Research output: Contribution to journalArticlepeer-review

Abstract

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 (Mr57000). 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.

Original languageEnglish (US)
Pages (from-to)2309-2314
Number of pages6
JournalBiochemistry
Volume25
Issue number9
DOIs
StatePublished - May 1986

ASJC Scopus subject areas

  • Biochemistry

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