Measurements of the proton motive force generated by cytochrome c oxidase from Bacillus subtilis in proteoliposomes and membrane vesicles.
Cytochrome c oxidase from Bacillus subtilis was reconstituted in liposomes and its energy-transducing properties were studied. The reconstitution procedure used included Ca2+-induced fusion of pre-formed membranes. The orientation of the enzyme in liposomes is influenced by the phospholipid composition of the membrane. Negatively charged phospholipids are essential for high oxidase activity and respiratory control. Analyses of the proteoliposomes by gel filtration, density gradient centrifugation and electron microscopy indicated a heterogeneity of the proteoliposomes with respect to size and respiratory control. Cytochrome c oxidase activity in the proteoliposomes resulted in the generation of a proton motive force, internally negative and alkaline. In the presence of the electron donor, ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c or ascorbate/phenazine methosulphate, the reconstituted enzyme generated an electrical potential of 84 mV which was increased by the addition of nigericin to 95 mV and a pH gradient of 32 mV which was increased by the addition of valinomycin to 39 mV. Similar results were obtained with beef-heart cytochrome c oxidase reconstituted in liposomes. The maximal proton motive force which could be generated, assuming no endogenous ion leakage, varied over 110-140 mV. From this the efficiency of energy transduction by cytochrome c oxidase was calculated to be 18-23%, indicating that the oxidase is an efficient proton-motive-force-generating system.
D001412:Bacillus subtilis / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004563:Electrochemistry; D003576:Electron Transport Complex IV / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000502:physiology*; D008564:Membrane Potentials; D008854:Microscopy, Electron; D010743:Phospholipids / Q000032:analysis; D011510:Proteolipids / Q000502:physiology; D011522:Protons; D013050:Spectrometry, Fluorescence
D008563:Membrane Lipids; D010743:Phospholipids; D011510:Proteolipids; D011522:Protons; C020485:proteoliposomes; D003576:Electron Transport Complex IV