Phosphorylation and kinetics of mammalian cytochrome c oxidase.
The influence of protein phosphorylation on the kinetics of cytochrome c oxidase was investigated by applying Western blotting, mass spectrometry, and kinetic measurements with an oxygen electrode. The isolated enzyme from bovine heart exhibited serine, threonine, and/or tyrosine phosphorylation in various subunits, except subunit I, by using phosphoamino acid-specific antibodies. The kinetics revealed slight inhibition of oxygen uptake in the presence of ATP, as compared with the presence of ADP. Mass spectrometry identified the phosphorylation of Ser-34 at subunit IV and Ser-4 and Thr-35 at subunit Va. Incubation of the isolated enzyme with protein kinase A, cAMP, and ATP resulted in serine and threonine phosphorylation of subunit I, which was correlated with sigmoidal inhibition kinetics in the presence of ATP. This allosteric ATP-inhibition of cytochrome c oxidase was also found in rat heart mitochondria, which had been rapidly prepared in the presence of protein phosphatase inhibitors. The isolated rat heart enzyme, prepared from the mitochondria by blue native gel electrophoresis, showed serine, threonine, and tyrosine phosphorylation of subunit I. It is concluded that the allosteric ATP-inhibition of cytochrome c oxidase, previously suggested to keep the mitochondrial membrane potential and thus the reactive oxygen species production in cells at low levels, occurs in living cells and is based on phosphorylation of cytochrome c oxidase subunit I.
Helling
Stefan
S
Medizinisches Proteom-Center, Funktionelle Proteomik, Ruhr-Universität Bochum, Germany.
Vogt
Sebastian
S
Rhiel
Annika
A
Ramzan
Rabia
R
Wen
Li
L
Marcus
Katrin
K
Kadenbach
Bernhard
B
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D002417:Cattle; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology / Q000648:ultrastructure; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D019000:Phosphotyrosine / Q000032:analysis; D051381:Rats; D012694:Serine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013912:Threonine / Q000378:metabolism
D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV