The effects of lipid fluidity on the rotational diffusion of complex I and complex III in reconstituted NADH-cytochrome c oxidoreductase.
NADH-ubiquinone oxidoreductase (Complex I) can be recombined with ubiquinol-cytochrome c oxidoreductase (Complex III) to reconstitute NADH-cytochrome c oxidoreductase. Two modes of interaction have been found. In one, the Complexes interact stoichiometrically in one to one molar ratios to give a binary Complex I-III unit. In the other, the kinetics of NADH-cytochrome c oxidoreductase are characteristic of 'Q-pool' behaviour seen in intact mitochondria and submitochondrial particles in which the Complexes need not interact directly but can do so via a pool of mobile ubiquinone. Stoichiometric behaviour is found when only boundary layer or annular lipid is present or the lipid is in the gel phase. The lipid is immobile on the ESR time scale and protein rotational diffusion, measured by saturation transfer ESR, is very slow. Q-pool behaviour is found when mobile extra-annular lipid phase is also present. Protein rotational diffusion is rapid and characteristic of a fully disaggregated state. We have also used freeze-fracture electron microscopy of reconstituted NADH-cytochrome c oxidoreductase to monitor protein aggregation and lateral phase separation of lipids and proteins under various conditions. We discuss our findings in relation to models for lateral interactions between respiratory chain enzymes.
D000818:Animals; D002417:Cattle; D003497:Cyclic N-Oxides; D003579:Cytochrome Reductases / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III; D005614:Freeze Fracturing; D008560:Membrane Fluidity; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000201:enzymology; D009097:Multienzyme Complexes / Q000378:metabolism*; D016660:NAD(P)H Dehydrogenase (Quinone); D009245:NADH Dehydrogenase / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011808:Quinone Reductases / Q000378:metabolism*; D013113:Spin Labels; D013696:Temperature
D003497:Cyclic N-Oxides; D009097:Multienzyme Complexes; D013113:Spin Labels; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl; D009247:NADH, NADPH Oxidoreductases; D003579:Cytochrome Reductases; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; D009245:NADH Dehydrogenase; D014450:Electron Transport Complex III