Role of phospholipids in respiratory cytochrome bc1 complex catalysis and supercomplex formation

Abstract

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 bc(1) complex and how disruption of a specific cardiolipin binding site in cytochrome c(1) alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc(1) complex was treated with phospholipase A(2). 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 b(L)) and from +113 to -2 mV (heme b(H)). 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 (CL(i)) in supercomplex formation by site-directed 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 bc(1) 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.

Cells Lacking Rieske Iron-Sulfur Protein Have a Reactive Oxygen Species-Associated Decrease in Respiratory Complexes I and IV

“…Respiratory complex and supercomplex instability has been attributed, in some instances, to low levels of phospholipids, such as cardiolipin (47,52,67). However, analysis of phospholipids by thin-layer chromatography did not reveal major differences between control and RISP KO cells (data not shown).…”

Arrangement of the Respiratory Chain Complexes in Saccharomyces cerevisiae Supercomplex III2IV2 Revealed by Single Particle Cryo-Electron Microscopy

“…Tightly bound CL in the crystal structure of the yeast complex III (CL denoted as CN3 in PDB 3CX5 and shown in Fig. 5) was demonstrated to be important for supercomplex stability (16). Although this tightly bound CL is necessary for supercomplex formation, it is probably not sufficient for stable supercomplex formation.…”

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

“…Whereas deletion of CL affects the stability of respiratory chain supercomplexes as well as of the TOM and TIM23 translocases, these protein complexes remain largely intact in PE-deficient mitochondria (49, 50, 53-58, 60 -62). CL associates with respiratory chain complexes (63,64) and stabilizes the interaction of the cytochrome c reductase (complex III) and complex IV via its negatively charged headgroup (65).…”