Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding

Abstract

Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance.cytochrome c oxidase | mass spectrometry | lipids

Role of conformational change and K-path ligands in controlling cytochrome c oxidase activity

“…In seminal studies on mammalian C c O, Kadenbach and co-workers demonstrated the regulation of activity and efficiency by many physiological ligands, including ATP, ADP, and thyroxin [1–4]. They further recognized that the enzyme was subject to phosphorylation, an early observation that is now supported by a great deal of data indicating numerous phosphorylation [5] and acetylation sites in mammalian C c O [6,7]. Equally significant was the discovery by the Kadenbach group that many isoforms of the nuclear encoded subunits existed and were differentially expressed in different tissues [8,9].…”

Cholesterol interaction sites on the transmembrane domain of the hedgehog signal transducer and Class F G protein-coupled receptor Smoothened

“…Despite these limitations, the approaches discussed have achieved significant success in identifying a range of lipid interaction sites on membrane proteins, controlled and tested against experimental data including mass spectrometry (Gupta et al, 2017;Liko et al, 2016;Yen et al, 2018), crystallographic (Arnarez et al, 2013a;Schmidt et al, 2013;Van Eerden et al, 2017;Zeppelin et al, 2018), NMR spectroscopy Hedger et al, 2016b), and mutational functional data (Hedger et al, 2016a;Hedger et al, 2015;Stansfeld et al, 2009).…”

Cytochrome c Oxidase: Insight into Functions from Studies of the Yeast S. cerevisiae Homologue

“…In yeast, only Cox5 (homologous to mammalian subunit IV) has isoforms but in mammalian CcOs isoforms of subunits IV, VIa, VIb, VIc, VIIa, VIIb and VIII have been found. Several subunits house sites that may become phosphorylated [Hüttemann et al, 2012], acetylated [Liko et al, 2016] or can bind nucleotides [Kadenbach and Hüttemann, 2015;Arnold, 2012]. Specific functions of some of these isoforms, their post-translational modifications and ligand binding sites have been suggested, though the supporting data in many cases remain inconsistent.…”