Liposome-mitochondrial inner membrane fusion. Lateral diffusion of integral electron transfer components.

The rates of specific catalytic events in the mitochondrial inner membrane were determined after enriching the membrane bilayer with exogenous phospholipid. Inner membranes were fused with soybean phospholipid, then separated into four buoyant-distinct fractions by sucrose density centrifugation. Analysis revealed a progressively increasing phospholipid to protein ratio from the least buoyant (30% phospholipidenriched) to the most buoyant (700% phospholipid-enriched) membrane fraction. This progressive increase was proportional to an increase in the surface area of the membrane bilayer and a decrease in the lateral density distribution of integral proteins observed as intramembrane particles by freeze-fracture electron microscopy. Thus, the average distance between integral proteins increased as the inner membrane bilayer was enriched with exogenous phospholipid. Electron transfer rates from NADH and succinate to cytochrome c as well as to oxygen decreased in proportion to the phospholipid enrichment and increased surface area of the membrane bilayer. A maximum decrease of 90% to 95% occurred in the most buoyant membranes. NADHand succinate-ubiquinone reductase activity decreased maximally by 60%, while ubiquinol-cytochrome c reductase activity decreased by 85%. No decrease occurred in the duroquinol oxidase activity of any of the phospholipid-enriched membrane fractions. Activities of the single enzymes, NADH dehydrogenase, cytochrome c oxidase, and ATPase increased with increasing phospholipid enrichment of the bilayer. These data identify a diffusion-limited step in electron transfer between the inner membrane dehydrogenases and cytochromes b c 1 and indicate that the dehydrogenases, ubiquinone, and cytochromes b c l are independent, diffusible membrane oxidation-reduction components.