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.

Fluorescence recovery after photobleaching was used to determine the diffusion coefficients of the oxidation-reduction (redox) components ubiquinone, complex III (cytochromes b-cl), cytochrome c, and complex IV (cytochrome oxidase) of the mitochondrial inner membrane. All redox components diffuse in two dimensions as common-pool electron carriers. Cytochrome c diffuses in two and three dimensions concomitantly, and its diffusion rate, unlike that of all other redox components, is modulated along with its activity by ionic strength. The diffusion coefficients established in this study reveal that the theoretical diffusion-controlled collision frequencies of all redox components are greater than their experimental maximum (uncoupled) turnover numbers. Since electron transport is slower than the theoretical limit set by the lateral diffusion of the redox components, ordered chains, assemblies, or aggregates of redox components are not necessary to account for electron transport. Rather, mitochondrial electron transport is diffusion coupled, consistent with a "random-collision model" for electron transport.A number of recent studies suggest that the oxidation-reduction (redox) components of the mitochondrial inner membrane are not arranged as an ordered macromolecular assembly or chain but are free to diffuse laterally and independently of one another in the membrane plane (1-5). These components include the dehydrogenase-containing complexes I and II, cytochrome b-cl complex III, cytochrome oxidase complex IV, cytochrome c, and ubiquinone. Implicit in these studies, and consistent with a "random-collision model," the rate of electron transfer between the membrane redox components may be diffusion coupled and, indeed, diffusion controlled (2).It is the purpose of this paper to report the rates of lateral diffusion for the inner membrane redox components as de-termined by fluorescence recovery after photobleaching and to evaluate such diffusion with respect to electron transport.Based on the diffusion coefficients established experimentally by our study, the calculated diffusion-controlled collision frequencies of all redox components are greater than their experimental maximum turnover numbers. Thus electron transfer between all redox components is diffusion coupled in the mitochondrial inner membrane, consistent with a random-collision model (2) (7), and purified mitoplasts (7) were washed in isolation medium diluted 1:7.5 (40 mOsm) to give spherical inner membranes (8), then washed twice with 0.15 M KCI/1 mM Hepes, pH 7.4, to remove native cytochrome c. Calcium-induced fusion of these inner membranes was carried out in a chamber x100 1Lm high formed between a glass coverslip and slide with Scotch double-stick tape shims. Twenty microliters of the spherical membranes (5 mg/ml) was added to the chamber from one side, and 20 ,ul of 10 mM CaC12/5 mM Hepes, pH 6.4, was added from the other side and then incubated at 35C for 7 min. Fusion was stopped by washing the chamber with 0.05 ml of 10 mM potassium phospha...

Regional myocardial ischemia was produced in anesthetized pigs by occluding the left anterior descending coronary artery. Mitochondria were prepared from both normally perfused and ischemic myocardium after 2 h of occlusion. Mitochondria from the ischemic area exhibited an 89% increase in cholesterol content from 32.7 +/- 1.9 (control) to 62.0 +/- 0.47 (ischemic) nmol/mg protein with no change in either total phospholipid content or in membrane fatty acid composition. This increase in mitochondrial membrane cholesterol was accompanied by an increase in membrane microviscosity as indicated by increased fluorescence polarization using the fluorescent membrane probe, 1,6-diphenyl-1,3,5-hexatriene. In these same experiments the Arrhenius plot discontinuity temperature of oligomycin-sensitive adenosinetriphosphatase (ATPase) activity fell from 20.0 to 14.2 degrees C. Our results suggest that, during the myocardial ischemic process in pigs, there is an intracellular redistribution of free cholesterol that produces a marked increase in mitochondrial membrane cholesterol content. This appears to produce an altered mitochondrial membrane lipid bilayer packing, resulting in increased membrane microviscosity and, possibly, altered inner membrane ATPase function. Intracellular cholesterol redistribution may thus contribute to the cell membrane damage that occurs during the myocardial ischemic process.

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