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 ATPinhibition 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.
Molecular & Cellular Proteomics 7:1714 -1724, 2008.Phosphorylation of mitochondrial proteins has become of general interest since the role of mitochondria in apoptosis and degenerative diseases became evident. During the past ten years many protein kinases and phosphatases, mostly known to occur outside of mitochondria, have also been identified in mitochondria or are translocated to mitochondria after activation (1-6). In addition, an increasing number of phosphorylated proteins, including subunits of complexes I-V of the mitochondrial oxidative phosphorylation system, have been identified (7-9). Of particular interest is the phosphorylation of cytochrome c oxidase (CcO) 1 , the terminal, and rate-limiting enzyme of the respiratory chain (complex IV) (10). CcO is composed of three mitochondrial DNA-encoded subunits, forming the catalytic core of the enzyme, and ten nuclear-encoded subunits with regulatory functions. The crystal structure of the bovine heart enzyme forms a dimer (11,12), and supercomplexes of CcO with complex III (cytochrome c reductase) and complex I (NADH dehydrogenase) have been identified in mitochondrial membranes (13-15). The complicated structure of the mammalian enzyme contrasts the bacterial CcO containing only 2-4 subunits (16,17). The additional subunits in eukaryotes are suggested to regulate CcO activity, either by binding effectors or by chemical modification, like glycosylation and phosphorylation. Ten high-affinity binding sites for ADP have been identified in the isolated bovine heart enzyme, seven of which are exchanged by ATP at ...