Cytochrome c oxidase from Paracoccus denitrificans: both hemes are located in subunit I.
The two-subunit cytochrome c oxidase from Paracoccus denitrificans has been sequentially digested with chymotrypsin and Staphylococcus aureus V8 protease. The smaller subunit of the enzyme (apparent Mr 32,000) was split into numerous peptides that were removed by anion-exchange HPLC. The larger subunit was only digested to a limited extent (from an apparent Mr 45,000 to Mr 43,000), and the spectral properties were preserved relative to the native enzyme (a reduced minus oxidized difference spectrum with maxima at 447 and 607 nm in the Soret and alpha region, respectively). As judged from CO-reduced spectra this proteolytically digested, one-fragment oxidase was found to contain an equal amount of cytochromes a and a3. The enzymatic activity with reduced cytochrome c as substrate in the presence of Triton X-100 proceeded with equal affinity (apparent Km = 0.5-1.0 microM) and with a Vmax of approximately 20% (40 s-1) of that found with the native enzyme (200 s-1). When the assay system was supplemented with soybean phospholipids, the Km became 2 microM for both enzymes and the Vmax became 730 and 170 s-1 for the native and the digested enzyme, respectively. Thus subunit I of P. denitrificans oxidase, and most probably of the other cytochrome c oxidases as well, contains both hemes and at least one Cu atom and has significant enzymatic activity.
Müller
M
M
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Schläpfer
B
B
Azzi
A
A
D002918:Chymotrypsin / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000032:analysis*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism
D046911:Macromolecular Substances; D003576:Electron Transport Complex IV; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase