Terminal Oxidases of Escherichia coli Aerobic Respiratory Chain 11 . PURIFICATION AND PROPERTIES OF CYTOCHROME bsss - d COMPLEX FROM CELLS GROWN

Cytochrome bSh8-d complex, a terminal oxidase in the respiratory chain of Escherichia coli K12 grown under the condition of limited oxygen, was purified to near homogeneity. The purified oxidase complex is composed of equimolar amounts of two polypeptides, with M, = 26,000 and 51,000, determined with gel eIectrophoresis in the presence of sodium dodecyl sulfate. It contains 12.3 nmol of protoheme, 9.54 nmol of cytochrome d, and 26.6 nmol of ironfmg of protein. The enzyme is a ”cytochrome bd-type oxidase,” which shows absorption peaks at 558 and 624 nm in the difference spectrum at 77 K. This oxidase combines with CO, and the Soret region of the CO difference spectrum at room temperature has a peak at 420 nm and troughs at 430 and 442 nm. The oxidation-reduction potentials of cytochrome bws and cytochrome d at pH 7.4 were estimated to be +10 mV and +240 mV, respectively. The cytochrome bSs8-d complex catalyzes the oxidation of ubiquinol-1, menadiol, and ascorbate in the pr sence of N,N,N‘,N’tetramethyl-p-phenylenediamine dihydrochloride. This oxidase activity was inhibited by the respiratory inhibitors piericidh A, KCN, and NaN3, but the cytochrome b5sa-d complex was less sensitive to the inhibitors than the cytochrome b66Z-0 complex. The K , values for oxygen of purified cytochrome b658-d complex and cytochrome b66z-0 complex were determined to be 0.38 and 2.9 PM, respectively. Formation of a membrane potential by the reconstituted cytochrome 655,-d complex in liposomes was observed with the fluorescent dye 3,3‘-dipropylthiocarbocyanine iodide on addition of ubiquinol1. This is the first indication that there is a coupling site in the terminal oxidase, which contains cytochrome d. Steady state kinetics of cytochromes in the membrane showed that these oxidase complexes branch at the site of ubiquinone-$ in the respiratory chain. From these and previous results, branched electron-carrying systems of the E. coli respiratory chain are proposed.