Formation of a membrane potential by reconstructed liposomes made with cytochrome b562-o complex, a terminal oxidase of Escherichia coli K12.

A terminal oxidase of the Escherichia coli K12 respiratory chain (cytochrome b562-o complex) was reconstituted into liposomes by freeze-thaw/sonication method. Formation of a membrane potential (-145 mV) by the reconstituted cytochrome b562-o complex was observed with the fluorescent dye 3,3'-dipropylthiodicarbocyanine iodide on addition of an artificial electron donor ubiquinol-1 or ascorbate-phenazine methosulfate. The membrane potential formed was inhibited by the protonophore uncouplers 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and carbonylcyanide m-chlorophenylhydrazone, and the inhibitors of the oxidase system zinc sulfate, potassium cyanide, and 2-n-heptyl-4-hydroxyquinoline-N-oxide. This is the first indication that there is a coupling site in an E. coli terminal oxidase, which consists of b-type cytochromes.