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Schlagwörter:
Mitochondria; Respiratory complex III; Cytochrome bc1 complex; Cardiolipin; Supercomplex
Zusammenfassung:
Specific protein–lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc1 complex and how disruption of a specific cardiolipin binding site in cytochrome c1 alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc1 complex was treated with phospholipase A2. The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from −52 to −82 mV (heme bL) and from +113 to −2 mV (heme bH). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CLi) in supercomplex formation by sitedirected mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc1 complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein–protein interaction.
