Spectroscopic and functional properties of subunit III-depleted cytochrome oxidase.
Beef heart cytochrome c oxidase has been depleted of subunit III by treatment with chymotrypsin. The removal of subunit III has been evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel fluorography of preparations of the oxidase labeled with [14C]dicyclohexylcarbodiimide prior to proteolysis. Removal of subunit III resulted in a perturbation of the visible spectrum of reduced cytochrome oxidase. Subunit III-depleted oxidase is spectroscopically very similar to the oxidase from Paracoccus denitrificans. When reconstituted into liposomes, the depleted enzyme still pumped protons in response to a pulse of reduced cytochrome c. The H+/e- stoichiometry averaged 0.5. Redox-linked proton translocation could be observed only when respiratory control ratios were higher than 3 and the reductant pulse was of a magnitude that allowed for no more than 5 turnovers of the oxidase.
Puettner
I
I
Carafoli
E
E
Malatesta
F
F
D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D003576:Electron Transport Complex IV; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology; D011522:Protons; D013053:Spectrophotometry
D046911:Macromolecular Substances; D011522:Protons; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003576:Electron Transport Complex IV; D002918:Chymotrypsin