Interactions of phospholipids with the mitochondrial cytochrome-c reductase studied by spin-label ESR and NMR spectroscopy.
Protein/phospholipid interactions in the solubilized mitochondrial ubihydroquinone:cytochrome-c oxidoreductase (bc1 complex) were studied by spin-label electron-spin resonance and by 31P-NMR spectroscopy. Spin-labelled phospholipids were employed to probe the relative binding affinities of a number of phospholipids with regard to the significance of phospholipids for the activity and stability of this multisubunit complex. The protein was titrated with spin-labelled cardiolipin (1,3-bisphosphatidyl-sn-glycerol) and with the spin-labelled analogues of PtdCho and PtdEtn, both of which have been shown recently to elicit a substantial increase in electron-transport activity [Schägger, H., Hagen, T., Roth, B., Brandt, U., Link, T. A. & von Jagow, G. (1990) Eur. J. Biochem. 190, 123-130]. A simplified distribution model showed that neutral phospholipids have much lower protein affinity than cardiolipin. In contrast to the transient weak lipid binding detected by spin-label electron-spin resonance, 31P NMR revealed a tightly bound cardiolipin portion, even after careful delipidation of the complex. Considerable line narrowing was observed after phospholipase A2 digestion of the bound cardiolipin, whereas addition of SDS resulted in complete release. Relative proportions and line widths of mobile and immobilized lipids were obtained by deconvoluting the partially overlapping signals. The current results are discussed with reference to similar findings with other mitochondrial membrane proteins. It is assumed that activation by neutral phospholipids reflects a generalized effect on the protein conformation. Cardiolipin binding is believed to be important for the structural integrity of the mitochondrial protein complexes.
Hayer-Hartl
M
M
Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiológie, Universität München, Federal Republic of Germany.
Schägger
H
H
von Jagow
G
G
Beyer
K
K
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002308:Cardiolipins / Q000378:metabolism; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008929:Mitochondria, Heart / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000378:metabolism*; D017830:Octoxynol; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D011092:Polyethylene Glycols; D013113:Spin Labels
D002308:Cardiolipins; D008823:Micelles; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D013113:Spin Labels; D011092:Polyethylene Glycols; D017830:Octoxynol; D009245:NADH Dehydrogenase; D010741:Phospholipases A; D054467:Phospholipases A2