title: Cardiolipin deficiency in Rhodobacter sphaeroides alters the lipid profile of membranes and of crystallized cytochrome oxidase, but structure and function are maintained.
abstract: Many recent studies highlight the importance of lipids in membrane proteins, including in the formation of well-ordered crystals. To examine the effect of changes in one lipid, cardiolipin, on the lipid profile and the production, function, and crystallization of an intrinsic membrane protein, cytochrome c oxidase, we mutated the cardiolipin synthase (cls) gene of Rhodobacter sphaeroides, causing a >90% reduction in cardiolipin content in vivo and selective changes in the abundances of other lipids. Under these conditions, a fully native cytochrome c oxidase (CcO) was produced, as indicated by its activity, spectral properties, and crystal characteristics. Analysis by MALDI tandem mass spectrometry (MS/MS) revealed that the cardiolipin level in CcO crystals, as in the membranes, was greatly decreased. Lipid species present in the crystals were directly analyzed for the first time using MS/MS, documenting their identities and fatty acid chain composition. The fatty acid content of cardiolipin in R. sphaeroides CcO (predominantly 18:1) differs from that in mammalian CcO (18:2). In contrast to the cardiolipin dependence of mammalian CcO activity, major depletion of cardiolipin in R. sphaeroides did not impact any aspect of CcO structure or behavior, suggesting a greater tolerance of interchange of cardiolipin with other lipids in this bacterial system.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, United States.
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
authors.forename: Banita
authors.initials: B
authors.lastname: Tamot
authors.forename: Carrie
authors.initials: C
authors.lastname: Hiser
authors.forename: Gavin E
authors.initials: GE
authors.lastname: Reid
authors.forename: Christoph
authors.initials: C
authors.lastname: Benning
authors.forename: Shelagh
authors.initials: S
authors.lastname: Ferguson-Miller
mesh_terms: D002308:Cardiolipins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D003460:Crystallization; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D050356:Lipid Metabolism / Q000502:physiology; D008563:Membrane Lipids / Q000096:biosynthesis / Q000737:chemistry* / Q000172:deficiency*; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D053719:Tandem Mass Spectrometry; D017855:Transferases (Other Substituted Phosphate Groups) / Q000172:deficiency / Q000235:genetics
chemical_list: D002308:Cardiolipins; D008563:Membrane Lipids; D008565:Membrane Proteins; D003576:Electron Transport Complex IV; D017855:Transferases (Other Substituted Phosphate Groups); C100091:cardiolipin synthetase