Bound cardiolipin is essential for cytochrome c oxidase proton translocation

Andrej Musatov, Neal C. Robinson

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

The proton pumping activity of bovine heart cytochrome c oxidase (CcO) is completely inhibited when all of the cardiolipin (CL) is removed from the enzyme to produce monomeric CcO containing only 11 subunits. Only dimeric enzyme containing all 13 subunits and 2-4 cardiolipin per CcO monomer exhibits a "normal" proton translocating stoichiometry of ∼1.0 H+ per/e- when reconstituted into phospholipid vesicles. These fully active proteoliposomes have high respiratory control ratios (RCR = 7-15) with 75-85% of the CcO oriented with the cytochrome c binding sites exposed to the external medium. In contrast, reconstitution of CL-free CcO results in low respiratory control ratios (RCR < 5) with the enzyme randomly oriented in the vesicles, i.e., ∼50 percent oriented with the cytochrome c binding site exposed on the outside of the vesicle. Addition of exogenous CL to the CL-free enzyme completely restores electron transport activity, but restoration of proton pumping activity does not occur. This is true whether CL is added to CL-free CcO prior to reconstitution into phospholipid vesicles, or whether CL is included in the phospholipid mixture that is used to form the vesicles. Another consequence of CL removal is the inability of the 11-subunit, CL-free enzyme to dimerize upon exposure to either cholate or the cholate/PC/PE/CL mixture used during proteoliposome formation (monomeric, 13-subunit, CL-containing CcO completely dimerizes under these conditions). Therefore, a major difference between reconstitution of CL-free and CL-containing CcO is the incorporation of monomeric, rather than dimeric CcO into the vesicles. We conclude that bound CL is necessary for proper insertion of CcO into phospholipid vesicles and normal proton translocation.

Original languageEnglish (US)
Pages (from-to)159-164
Number of pages6
JournalBiochimie
Volume105
DOIs
StatePublished - Oct 2014

Keywords

  • Cardiolipin
  • Cytochrome c oxidase
  • Liposomes
  • Proton pumping

ASJC Scopus subject areas

  • Biochemistry

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