coupling sites, is simply the negative of PMF(import). The spontaneity of proton import (from the P to the N side) is universal in all bioenergetic membranes. This fact was not recognized before the 1990s, because the chloroplast thylakoid lumen was interpreted as an interior phase, but in fact it is topologically equivalent to the exterior of the chloroplast. Azzone et al. stressed that the inside phase (N side of the membrane) is the bacterial cytoplasm, mitochondrial matrix, or chloroplast stroma; the outside (P) side is the bacterial periplasmic space, mitochondrial intermembrane space, or chloroplast lumen. Furthermore, 3D tomography of the mitochondrial inner membrane shows its extensive invaginations to be stacked, similar to thylakoid disks; hence the mitochondrial intermembrane space is topologically quite similar to the chloroplast lumen.: The energy expressed here as Gibbs free energy, electrochemical proton gradient, or proton-motive force (PMF), is a combination of two gradients across the membrane: the concentration gradient (via ) and electric potential gradient . When a system reaches equilibrium, ; nevertheless, the concentrations on either side of the membrane need not be equal. Spontaneous movement across the potential membrane is determined by both concentration and electric potential gradients. The molar Gibbs free energy of ATP synthesis is also called phosphorylation potential. The equilibrium concentration ratio can be calculated by comparing and , for example in case of the mammalian mitochondrion: H+ / ATP = ΔGp / (Δp / 10.4 kJ·mol−1/mV) = 40.2 kJ·mol−1 / (173.5 mV / 10.4 kJ·mol−1/mV) = 40.2 / 16.7 = 2.4. The actual ratio of the proton-binding c-subunit to the ATP-synthesizing beta-subunit copy numbers is 8/3 = 2.67, showing that under these conditions, the mitochondrion functions at 90% (2.4/2.67) efficiency. In fact, the thermodynamic efficiency is mostly lower in eukaryotic cells because ATP must be exported from the matrix to the cytoplasm, and ADP and phosphate must be imported from the cytoplasm. This "costs" one "extra" proton import per ATP, hence the actual efficiency is only 65% (= 2.4/3.67). In mitochondria The complete breakdown of glucose releasing its energy is called cellular respiration. The last steps of this process occur in mitochondria. The reduced molecules NADH and FADH2 are generated by the Krebs cycle, glycolysis, and pyruvate processing. These molecules pass electrons to an electron transport chain, which releases the energy of oxygen to create a proton gradient across the inner mitochondrial membrane. ATP synthase then uses the energy