By means of a new "quick-sampling" method, micropellcts of mouse liver mitochondria were rapidly prepared for electron microscopy during the recording of steady state metabolism. Reversible ultrastructural changes were found to accompany change in metabolic steady states. The most dramatic reversible ultrastructural change occurs when ADP is added to systems in which only phosphate acceptor is deficient, i.e., during the State IV to State III transition as defined by Chance and Williams. After 15 rain in State IV, mitochondria display an "orthodox" ultrastructural appearance as is usually observed after fixation within intact tissue. On transition to State III, a dramatic change in the manner of folding of the inner membrane takes place. In addition, the electron opacity of the matrix increases as the volumc of the matrix decrcases, but total mitochondrial volume does not appear to change during this transition. This conformation is called "condensed." Isolated mitochondria were found to oscillate bctwecn the orthodox and condensed conformations during reversible transitions between State III and State IV. Various significant ultrastructural changes in mitochondria also occur during transitions in other functional states, e.g., when substrate or substratc and acceptor is made limiting. Internal structural flexibility is discussed with respect to structural and functional integrity of isolated mitochondria. Reversible changcs in the manner of folding of the inner membrane and in the manner of packing of small granules in thc matrix as respiration is activated by ADP represent an ultrastructural basis for metabolically linked mechanical activity in tightly coupled mitochondria.

This review focuses on our studies over the past ten years which reveal that the mitochondrial inner membrane is a fluid-state rather than a solid-state membrane and that all membrane proteins and redox components which catalyze electron transport and ATP synthesis are in constant and independent diffusional motion. The studies reviewed represent the experimental basis for the random collision model of electron transport. We present five fundamental postulates upon which the random collision model of mitochondrial electron transport is founded: All redox components are independent lateral diffusants; Cytochrome c diffuses primarily in three dimensions; Electron transport is a diffusion-coupled kinetic process; Electron transport is a multicollisional, obstructed, long-range diffusional process; The rates of diffusion of the redox components have a direct influence on the overall kinetic process of electron transport and can be rate limiting, as in diffusion control. The experimental rationales and the results obtained in testing each of the five postulates of the random collision model are presented. In addition, we offer the basic concepts, criteria and experimental strategies that we believe are essential in considering the significance of the relationship between diffusion and electron transport. Finally, we critically explore and assess other contemporary studies on the diffusion of inner membrane components related to electron transport including studies on: rotational diffusion, immobile fractions, complex formation, dynamic aggregates, and rates of diffusion. Review of all available data confirms the random collision model and no data appear to exist that contravene it. It is concluded that mitochondrial electron transport is a diffusion-based random collision process and that diffusion has an integral and controlling affect on electron transport.

Isolated mitochondria are capable of undergoing dramatic reversible ultrastructural transformations between a condensed and an orthodox conformation. These two conformations are the extremes in ultrastructural organization between which structually and functionally intact mitochondria transform during reversible respiratory cycles. It has been found that electron transport is required for the condensed-to-orthodox ultrastructural transformation which occurs in mitochondria under State IV conditions, i.e., under conditions in which exogenous substrate is present and ADP is deficient. Inhibition of State IV electron transport at the cyanide-, antimycin A-, or Amytal-sensitive sites in the respiratory chain results in inhibition of this transformation. Resumption of electron transport in initially inhibited mitochondrial systems, initiated by channeling electrons through pathways which bypass the inhibited sites, results in resumption of the ultrastructural transformation. The condensed-toorthodox transformation is DNP insensitive and, therefore, does not require participation of the coupling enzymes of the energy-transfer pathway. It is concluded that this ultrastructural transformation is manifest by the conversion of the chemical energy of electron transport directly into mechanical work. The reversed ultrastructural transformation, i.e., orthodoxto-condensed, which occurs during ADP-activated State III electron transport, is inhibited by DNP and parallels suppression of acceptor control and oxidative phosphorylation. Mechanochemical ultrastructural transformation as a basis for energy transfer in mitochondria is considered with respect to the results presented.

Previous investigations in this laboratory have established that isolated liver mitochondria undergo reversible ultrastructural transformations during change in respiratory state.1' 2 Major transformations in the manner of folding of the inner membrane and in the volume and electron density of the matrix were observed.2 These and more recent observations with inhibitors of respiration and phosphorylation suggested that the ultrastructural transformations were manifestations of the transduction of oxidation-reduction energy into mechanical or conformational energy.3 It was postulated that conformational energy generated by electron transport is the immediate source of energy for the synthesis of ATP during oxidative phosphorylation.3 Other workers studying heart mitochondria recently summarized similar suggestions.4In this paper the ultrastructure of the condensed and the orthodox conformations,3 which represent the low-and high-energy states of isolated rat liver mitochondria, respectively, are examined by high-res~lution electron microscopy after chemical fixation with osmium tetroxide and after physical fixation by freezeetching. The results indicate that the matrix of intact isolated mitochondria is a structured, reticular network of protein which undergoes change in its geometric arrangement dependent on respiratory state. This matrix network is physically continuous with the inner mitochondrial membrane. In addition, specific sites of physical contact are observed between the inner and the outer membrane of isolated mitochondria.Methods.-Mitochondria were isolated from rat liver essentially according to the method of Schneider.5 Their respiration rate was monitored with a Clark oxygen electrode as previously described.2 For chemical fixation, osmotically adjusted, phosphatebuffered, 2% osmium tetroxide (pH 7.4) was used on mitochondria in different states of respiration as previously described.2To prepare mitochondria for freeze-etching, 1.0-,yl samples of dilute mitochondria (18 mg protein per ml) and of concentrated mitochondrial micropellets (0.18 mg protein centrifuged at 15,000 g for 30 see) were placed on gold-nickel specimen disks. Glycerol and dimethyl sulfoxide were found to depress both acceptor control and oxidative phosphorylation and therefore were not used in this study to compress the crystallization interval of the specimens during freezing. Rapid vitrification was ensured by freezing all samples at -210'C against a surface of solid nitrogen. Fracturing, vacuum sublimation, and carbon shadowing were carried out at -1000C at '-'10-7 Torr in a Balzers BA 360 freeze-microtome high-vacuum plant.6 Thin carbon replicas (50 A thick) were found to result in finer granularity (resolution 20 A), although more transparent than platinum-carbon replicas.Electron micrographs were taken on Kodak 31/4 X 4-in. contrast plates at initial magnifications of 15,000 to 50,000, with an RCA 3G electron microscope operated at 100 kv and equipped with an anticontamination cold trap and a double condenser.Results.-Chem...