Terminal oxidases of Escherichia coli aerobic respiratory chain. II. Purification and properties of cytochrome b558-d complex from cells grown with limited oxygen and evidence of branched electron-carrying systems.

Abstract

Cytochrome b558-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 Mr = 26,000 and 51,000, determined with gel electrophoresis 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 iron/mg 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 b558 and cytochrome d at pH 7.4 were estimated to be +10 mV and +240 mV, respectively. The cytochrome b558-d complex catalyzes the oxidation of ubiquinol-1, menadiol, and ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. This oxidase activity was inhibited by the respiratory inhibitors piericidin A, KCN, and NaN3, but the cytochrome b558-d complex was less sensitive to the inhibitors than the cytochrome b562-o complex. The Km values for oxygen of purified cytochrome b558-d complex and cytochrome b562-o complex were determined to be 0.38 and 2.9 microM, respectively. Formation of a membrane potential by the reconstituted cytochrome b558-d complex in liposomes was observed with the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide on addition of ubiquinol-1. 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-8 in the respiratory chain. From these and previous results, branched electron-carrying systems of the E. coli respiratory chain are proposed.