Two-dimensional diffusion of F 1F 0-ATP synthase and ADP/ATP translocator. Testing a hypothesis for ATP synthesis in the mitochondrial inner membrane

Abstract

We report here the first experimentally determined lateral diffusion coefficients of the F 1F 0-ATP synthase and the ADP/ATP translocator in isolated inner membranes of rat liver mitochondria. Rabbit IgG developed against the F 1F 0-ATP synthase isolated from rat liver mitochondria was determined to be immunospecific for the synthase subunits, notably the α-β doublet, γ and δ subunits of F 1 and subunits two, three and four of F 0. This IgG, conjugated with lissamine-rhodamine, was used as a fluorescent probe to monitor the diffusion of the synthase in the membrane. IgG to cytochrome bc 1 complex, prepared and labeled similarly, was used as a fluorescent probe for diffusion of this redox component. Eosin maleimide was determined to specifically label the ADP/ATP translocator in the isolated inner membrane and was used as a specific probe for the diffusion of the translocator. Using fluorescence recovery after photobleaching, the experimental average lateral diffusion coefficient of the F 1F 0-ATP synthase was determined to be 8.4·10 −10 cm 2/s or twice that of cytochrome bc 1 complex while the diffusion coefficient of the ADP/ATP translocator was 1.7·10 −9 cm 2/s or four times that of cytochrome bc 1 complex suggesting that all three components are independent two-dimensional diffusants. Using these diffusion coefficients and applying a number of basic assumptions, we calculated the theoretical two-dimensional diffusion-controlled collision frequencies and derived collision efficiencies (protons transferred per collision) between each of the three proton-transferring redox complexes and both the F 1F 0-ATP synthase and ADP/ATP translocator by treating the redox components as proton donors and the synthase and translocator as proton acceptors. These collision efficiencies support the physical possibility of a diffusion-based, random collision process of proton transfer and ATP synthesis in the mitochondrial inner membrane.