The role of subunit III in bovine cytochrome c oxidase. Comparison between native, subunit III-depleted and Paracoccus denitrificans enzymes.
In order to obtain information on the role of subunit III in the function and aggregation state of cytochrome c oxidase, the kinetics of ferrocytochrome c oxidation by the bovine cytochrome c oxidase depleted of its subunit III were studied and compared with those of the oxidase isolated from P. denitrificans which contains only two subunits. The aggregation state of both enzymes dispersed in dodecyl maltoside was also compared. The two-subunit oxidase from P. denitrificans gave linear Eadie-Hofstee plots and the enzyme resulted to be monomeric (Mr = 82 000) both, in gel filtration and sucrose gradient centrifugation studies. The bovine heart subunit III depleted enzyme, under conditions when the P. denitrificans cytochrome c oxidase was in the form of monomers, was found to be dimeric by sucrose gradient centrifugation analysis. At lower enzyme concentrations monomers were, however, detected by gel filtration. Depletion of subunit III was accompanied by the loss of small polypeptides (VIa, VIb and VIIa) and of almost all phospholipid (1-2 molecules were left per molecule of enzyme). The electron-transfer activity of the subunit III-depleted enzyme showed a monophasic Eadie-Hofstee plot, which upon addition of phospholipids became non-linear, similar to that of the control bovine cytochrome c oxidase. One of the roles of subunit III may be that of stabilising the dimers of cytochrome c oxidase. Lack of this subunit and loss of phospholipid is accompanied by a change in the kinetics of electron transfer, which might be the consequence of enzyme monomerisation.
Nałeçz
K A
KA
Bolli
R
R
Ludwig
B
B
Azzi
A
A
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D010231:Paracoccus denitrificans / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation; D011522:Protons; D013053:Spectrophotometry
D010743:Phospholipids; D011522:Protons; D003576:Electron Transport Complex IV