Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity.
Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.
Müller
M
M
Institut für Biochemie und Molekularbiologie der Universität Bern, Switzerland.
Schlapfer
B
B
Azzi
A
A
D000345:Affinity Labels; D002918:Chymotrypsin / Q000378:metabolism; D003545:Cysteine / Q000032:analysis; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D017830:Octoxynol; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011092:Polyethylene Glycols; D011189:Potassium Chloride / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism; D013053:Spectrophotometry
D000345:Affinity Labels; D046911:Macromolecular Substances; D011092:Polyethylene Glycols; D011189:Potassium Chloride; C033531:eosin maleimide; D017830:Octoxynol; D003576:Electron Transport Complex IV; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D003545:Cysteine; D004801:Eosine Yellowish-(YS)