Properties of the Two Terminal Oxidases of Escherichia coli

Proton translocation coupled to oxidation of ubiquinol by O₂ was studied in spheroplasts of two mutant strains of Escherichia coli, one of which expresses cytochrome d, but not cytochrome bo, and the other expressing only the latter. O₂ pulse experiments revealed that cytochrome d catalyzes separation of the protons and electrons of ubiquinol oxidation but is not a proton pump. In contrast, cytochrome bo functions as a proton pump in addition to separating the charges of quinol oxidation. E. coli membranes and isolated cytochrome bo lack the Cu_A center typical of cytochrome c oxidase, and the isolated enzyme contains only lCu/2Fe. Optical spectra indicate that high-spin heme o contributes <10% to the reduced minus oxidized 560-nm band of the enzyme. Pyridine hemochrome spectra suggest that the hemes of cytochrome bo are not protohemes. Proteoliposomes with cytochrome bo exhibited good respiratory control, but H⁺/e⁻ during quinol oxidation was only 0.3-0.7. This was attributed to an “inside out” orientation of a significant fraction of the enzyme. Possible metabolic benefits of expressing both cytochromes bo and d in E. coli are discussed.