158Starting from 1961, two models for transforma tion of the energy of oxidation reactions in mito chondria have been discussed in the literature. The model of Mitchell [1] implies that the multienzyme phosphorylation system can work in a dissociated form, while the other model (by Williams) considers a supercomplex [2].We have earlier demonstrated that the phosphory lation system is able to function in both modes, corre sponding to two structural states of mitochondria [3]. The operation modes of the phosphorylating system are provided by the system of mitochondrial volume regulation [4,5].In hypotonic media under conditions of low amplitude mitochondrial swelling, the Williams model considers protons (unstably bound to the membrane supercomplex as Brønsted acids) as a substrate for ATP synthase.In 1976, we demonstrated the possibility to use a Brønsted acid as a substrate for ATP synthase using a model system. In these experiments, ATP synthase was sorbed at the octane-water interface. In this sys tem, ATP synthesis was observed in the presence of ADP and phosphate, and the gradient of a Brønsted acid (pentachlorophenol) at the octane-water inter face was used as a source of energy; the gradient was created by adding pentachlorophenol to the octane phase [6].On the bilayer membrane, we have found the cata lysts that selectively accelerate dissociation of Brøn sted acids [7]. This allowed us to find [8] that a non equilibrium fraction of Brønsted acids with excess free energy was formed on the surface of mitoplasts (mito chondria without the outer membrane), provided that H + pumps function.The goal of this work was to prove the formation of Brønsted acids during ATP synthesis on the mito chondrial membranes.A tightly joining of the cristae (Figs. 1 and 2) and, correspondingly, the joining of the outer and inner membranes should be expected. In this process, a two membrane structure of mitoplasts is formed of the outer and inner membranes. Since the outer mem brane is well permeable ("transparent") for hydrogen ions in course of proton pumping, this structure should function as an integral whole interacting with hydrogen ions, possessing excess free energy (accord ing to the scheme in Fig. 1b).We have earlier demonstrated that the measure ment of surface ζ potential (using a Zetasizer Nano ZS device) of mitoplasts can be efficiently used for detecting formation of nonequilibrium Brønsted acids in functional states [8].To selectively accelerate dissociation of mem brane bound Brønsted acids, possessing excess free energy, we increased the concentration of catalyst (HEPES) [7].The measurements were performed in the media with a tonicity of 350 and 120 mOsm. In this experi ment, we compared the measured ζ potential on the surface of functional mitochondria during ATP syn thesis under isotonic and hypotonic conditions. The experiments conducted under hypotonic conditions have demonstrated (Table 1) that the changes in ζ potential on the mitochondrial surface regularly cor relate with the changes in ζ potential on t...

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