Purification and properties of cytochrome b556 in the respiratory chain of aerobically grown Escherichia coli K12

Abstract

Cytochrome b556, a major component of type b cytochromes in the respiratory chain of aerobically grown Escherichia coli, was purified to near homogeneity. It was solubilized from cytoplasmic membranes by treatment with Sarkosyl/cholate mixture and purified by gel filtration on Sephadex G-200. The purified cytochrome b556 is an oligomer composed of identical polypeptides, with a molecular weight of 17,500, determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains equimolar amounts of heme and polypeptide but no detectable non-heme iron, phospholipid, or dehydrogenase. Its isoelectric point was determined to be 8.5. The cytochrome b556 is highly hydrophobic in its amino acid composition and does not contain any half-cystine residues. The purified cytochrome b556 is spectrophotometrically pure and the alpha absorption peak in its difference spectrum at 77 K is at 556 nm. The molar extinction coefficient of cytochrome b556 was determined as 22.8 cm-1 mM-1. Its oxidation-reduction potential was found to be -45 mV. It could be reduced by D-lactate dehydrogenase of E. coli in the presence of menadione.