Abstract
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.
Highlights
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
Ten high-affinity binding sites for ADP have been identified in the isolated bovine heart enzyme, seven of which are exchanged by ATP at high ATP/ADP ratios [18, 19]
Rapid isolation of mitochondria from rat heart in the presence of various protein phosphatase inhibitors resulted in cytochrome c oxidase (CcO) kinetics with allosteric ATP-inhibition and phosphorylation of subunit I at serine, threonine, and tyrosine
Summary
CcO, cytochrome c oxidase; ACN, acetonitril; FA, formic acid; ESI, electrospray ionization; CID, collisioninduced dissociation; ETD, electron transfer dissociation; PKA, protein kinase A; MS, mass spectrometry; ROS, reactive oxygen species. In the isolated bovine heart enzyme, three phosphorylated amino acids have been identified by mass spectrometry, i.e. Ser-34 of subunit IV and Ser-4 and Thr-35 of subunit Va. Incubation of isolated CcO with PKA, cAMP, and ATP resulted in phosphorylation of CcO subunit I at serine and threonine, accompanied by induction of allosteric ATP-inhibition. Rapid isolation of mitochondria from rat heart in the presence of various protein phosphatase inhibitors resulted in CcO kinetics with allosteric ATP-inhibition and phosphorylation of subunit I at serine, threonine, and tyrosine. The results indicate that the allosteric ATP-inhibition of CcO is present under in vivo conditions, but is immediately lost during stress, such as death of an animal
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