Properties of the two terminal oxidases of Escherichia coli

• Published in: Biochemistry (Easton) Vol. 30; no. 16; pp. 3936 - 3942
• Main Authors: Puustinen, Anne, Finel, Moshe, Haltia, Tuomas, Gennis, Robert B, Wikstrom, Marten
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 23.04.1991
• Subjects: 550200 - Biochemistry; ABSORPTION SPECTRA; BACTERIA; BASIC BIOLOGICAL SCIENCES; BIOCHEMISTRY; Biological and medical sciences; Cardiolipins; CELL CONSTITUENTS; CELL MEMBRANES; Cell structures and functions; CHEMISTRY; CYTOCHROME OXIDASE; CYTOCHROMES; ELECTRON SPECTRA; Electron Spin Resonance Spectroscopy; Electron Transport Complex IV - isolation & purification; Electron Transport Complex IV - metabolism; ENZYMES; ESCHERICHIA COLI; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; HAEM DEHYDROGENASES; Hydrogen-Ion Concentration; Kinetics; LIPOSOMES; MEMBRANES; MICROORGANISMS; Mitochondria and cell respiration; Molecular and cellular biology; ORGANIC COMPOUNDS; OXIDOREDUCTASES; Oxygen Consumption; PIGMENTS; PROTEINS; Proteolipids - metabolism; REDOX POTENTIAL; SPECTRA; Spectrophotometry; Spheroplasts - enzymology; Cytochrome; Purification; Escherichia coli; EPR spectrometry; Proton transfer; Liposome; Enzymatic activity; Polarography; Reconstituted system; Reaction mechanism; Bacteria; Chemical composition; Mutation; Enterobacteriaceae
• DOI: 10.1021/bi00230a019
• ISSN: 0006-2960

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 CuA center typical of cytochrome c oxidase, and the isolated enzyme contains only 1Cu/2Fe. Optical spectra indicate that high-spin heme o contributes less than 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.