Studies on the role of the oligomeric state and subunit III of cytochrome oxidase in proton translocation.
Anion-exchange fast protein liquid chromatography in the presence of lauryldimethylamine N-oxide (LDAO) was introduced to separate cytochrome oxidase into different complexes that either did or did not contain subunit III. Both kinds of enzyme complex exhibited H+ translocation after reconstitution into phospholipid vesicles, but with a significantly (approx. 50-60%) reduced H+/e- ratio as compared with unchromatographed enzyme. The anion-exchange FPLC fractions of the enzyme (with or without subunit III) sedimented more slowly than the control enzyme upon sucrose gradient centrifugation in the presence of cholate and a high potassium phosphate concentration. When the control enzyme was subjected to the sucrose gradient centrifugation in the presence of LDAO or Triton X-100, instead of cholate, one band containing all subunits was observed, which sedimented slowly like the FPLC fractions. Transfer of this band to cholate medium, and reapplication on the sucrose gradient (with cholate), yielded both a slow- and a fast-migrating band after centrifugation. Enzyme complexes that sedimented slowly or rapidly in the sucrose gradients revealed longer and shorter elution times, respectively, in gel filtration FPLC. This suggests that these complexes corresponds to monomers and dimers of cytochrome oxidase. Solubilization of proteoliposomes and subsequent sucrose gradient centrifugation in cholate yielded one fast-migrating band for the untreated enzyme, but both a fast- and a slow-migrating band for the anion-exchange FPLC-treated enzyme, which was exclusively slow-migrating before reconstitution into liposomes. It is suggested that dimerisation of monomeric cytochrome oxidase may be favoured when the enzyme encounters a membranous milieu, and that the dimeric structure might be necessary for proton translocation.
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology; D011522:Protons; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D011522:Protons; D003576:Electron Transport Complex IV