Multiple conformations of physiological membrane-bound cytochrome c.

One-tenth of cytochrome c (cyt c) remains bound to the inner mitochondrial membrane (IMM) at physiological ionic strength (I; i.e. , I approximately 150 mM), exhibiting decreased electron transport (ET) activity. We now show that this form of membrane-bound cyt c (MB-cyt c) can be obtained in vitro and that binding to membranes at low I generates an additional conformation with higher ET activity. This low I bound form of MB-cyt c (MBL-cyt c) exhibited intrinsic ET rates similar to those of electrostatically bound cyt c (EB-cyt c). The ET activity of IMM-bound MB-cyt c approached slowly that of MBL-cyt c or EB-cyt c, suggesting that MB-cyt c converts to MBL-cyt c while bound to IMM. When maintained at physiological I, both forms of MB-cyt c were released from the membrane, indicating that they convert to an EB-cyt c-like form. This process may be very dynamic in cellular mitochondria, as binding and release for both MB-cyt c forms increased considerably with temperature. I-Dependent binding of MB-cyt c does not require IMM, and it can be reproduced using large or small unilamellar vesicles (SUV). Using SUV-cyt c complexes, we characterized the secondary structure of MB-cyt c and MBL-cyt c by circular dichroism. Conformational analysis revealed that cyt c binding as MB-cyt c decreases its alpha-helical content (70-79%) and increases its beta-sheet up to 135%. The secondary structure of MBL-cyt c was similar to that of EB-cyt c and soluble cyt c, with a modest increase in beta-sheet. Taken together, our experiments suggest that physiological cyt c exists in soluble and membrane-bound conformations with similar ET activity, which may exchange very rapidly, and that soluble hydrophilic proteins can bind transiently to biomembranes.

Novel fluorescence membrane fusion assays reveal GTP-dependent fusogenic properties of outer mitochondrial membrane-derived proteins.

We have shown that fusion of small unilamellar vesicles (SUV) with outer mitochondrial membranes occurs at physiological pH [Cortese et al., 1991, J. Cell Biol., Vol. 113, 1331-1340]. The proteins driving this process could be involved in mitochondrial membrane fusion, which is presently poorly understood. In this study, we release from rat liver mitochondria a soluble protein fraction (SF) that increases fusion at neutral pH measured by membrane fusion assays (MFAs). Since this fusogenic activity was specifically enhanced by GTP, we separate SF by GTP affinity chromatography into: i) a flow-through subfraction (G1) containing numerous proteins with low GTP affinity; and ii) a subfraction (G2) which may contain GTP-binding proteins. A novel array of MFAs is developed to study the fusogenic properties of these fractions, measuring the merging of membranes (membrane-mixing) or the mixing of intravesicular aqueous contents (content-mixing). The MFAs use: a) SUV/large unilamellar vesicles, lacking mitochondrial membranes; b) SUV/mitochondria, reconstituting membrane-mitochondrial interactions; and c) mitochondria/mitochondria, mimicking mitochondrial fusion. The results indicate that: i) G1 contains GTP-independent, in vitro fusogenic proteins that are not sufficient to induce mitochondrial fusion; and ii) G2 contains GTP-dependent proteins that stimulate mitochondrial fusion at neutral pH. The MFAs described here could be used to monitor the isolation of active proteins from these subfractions and to define the mechanism of intermitochondrial membrane fusion.

Persistence of cytochrome c binding to membranes at physiological mitochondrial intermembrane space ionic strength.

We have shown that cytochrome c (cyt c) diffuses primarily in three dimensions in the intermembrane space (IMS) of intact mitochondria at physiological ionic strength (I). Recently, we found that a small percentage (11.2 +/- 2.1%) of endogenous cyt c remains bound to inner mitochondrial membranes (IMM) at high, physiological I (I = 150 mM), even after extensive washing with solutions at physiological I, overnight dialysis, changes in medium osmolarity, or further purification of IMM at high I using self-generating Percoll gradients. Measurements of heme c/heme a ratios, and electron transport (ET) reactions in which cyt c participates, confirmed the presence of a low amount of this I-resistant, membrane-bound form of cyt c (MB-cyt c), that had one third of the ET activity of electrostatically-bound cyt c (EB-cyt c), and which could not account for maximal ET rates. The amount of MB-cyt c was significantly increased above endogenous MB-cyt c by exposing KCl-washed IMM to increasing concentrations of exogenous cyt c. Also, subjecting large unilamellar vesicles (LUV) to successive cycles of cyt c binding/high I KCl-washes gave progressive increases in MB-cyt c. These protocols allowed in vitro characterization of MB-cyt c. The I at which binding takes place affects the affinity of cyt c for membranes, and oxidized cyt c had a greater intrinsic affinity for IMM or SUV than reduced cyt c. MB-cyt c appears to be bound partially by hydrophobic interactions since MB-cyt c was detected on negatively charged (asolectin) LUV and also on neutral, zwitterionic (phosphatidylcholine) LUV at high I. Consistent with the concentration-dependent changes in MB-cyt c, decreasing the IMS-volume of intact mitochondria (i.e., increasing th endogenous IMS-cyt c concentration) by metabolic or osmotic means increased the amount of MB-cyt c. After cyt c was delivered into the IMS by liposome-mediated low pH-induced fusion, resonance energy transfer showed a time-dependent cyt c-membrane proximity which was consistent with slow exchange of soluble IMS-entrapped cyt c molecules with a population bound to membranes at I = 150 mM. We conclude that, even though the majority of functional IMS-cyt c diffuses in three dimensions, a small portion remains firmly bound on the surface of the IMM under I conditions that are physiological for intact mitochondria. The occurrence of MB-cyt c may reflect an intrinsic conformational flexibility in cyt c, that allows a degree of membrane penetration and the formation of hydrophobic interactions which stabilize the membrane-bound form. The persistence of cyt c-membrane interactions under physiological I conditions indicates that cyt c-mediated ET in the IMS involves both fast (3D-diffusion) and slow (2D-diffusion) pathways for electron transfer.

Motional dynamics of functional cytochrome c delivered by low pH fusion into the intermembrane space of intact mitochondria.

We have investigated the motional dynamics of cytochrome c in the intact, functional rat liver mitochondrion. To do this, functional, FITC-cytochrome c (fluorescein isothiocyanate monoderivatized cytochrome c) was incorporated into the intermembrane space (IMS) of intact mitochondria through encapsulation of cytochrome c into asolectin liposomes followed by low pH-induced fusion of the liposomes with the outer membranes of the mitochondria. A cytochrome c controlled enrichment of between 15%-50% (1800-7200 molecules incorporated per mitochondrion) was obtained. All cytochrome c incorporated, regardless of the quantity, participated in the function of electron transport, indicative of a functional, independent random diffusant. Resonance energy transfer was determined from the IMS-entrapped functional FITC-cytochrome c to octadecylrhodamine B incorporated into the mitochondrial membranes. Resonance energy transfer from FITC-cytochrome c to octadecylrhodamine B in isolated inner or outer mitochondrial membranes (IMM and OMM, respectively) was also measured. We found substantial differences in the effects of ionic strength (I) on the proximity of cytochrome c to isolated IMM and OMM. Interactions with isolated IMM were very dynamic, i.e., very I-dependent, and cytochrome c binding to IMM was significant only at very low I. I-dependent interactions of cytochrome c with isolated OMM were less I-dependent than those for the IMM. However, FITC-cytochrome c was essentially released from IMM and OMM at physiological I. The proximity of FITC-cytochrome c to each mitochondrial membrane after its incorporation into the IMS of intact mitochondria in the condensed configuration was estimated at different external, bulk I using: (a) resonance energy transfer from IMS-entrapped FITC-cytochrome c to octadecylrhodamine B-label evenly distributed in both mitochondrial membranes; and (b) resonance energy transfer from IMS-entrapped FITC-cytochrome c to octadecylrhodamine B-label concentrated in the OMM. Resonance energy transfer showed that the average distance between cytochrome c and the two IMS-membrane surfaces increased with increasing IMS-I, approaching a maximal measurable distance of 85 A at 150 mM I. This result is consistent with a dissociation of FITC-cytochrome c and both membranes of intact mitochondria at physiological I, i.e., when the activity of cytochrome c in electron transport is highest. Our findings reveal a primarily three-dimensional diffusion mode for IMS-cytochrome c during its function in electron transport in intact mitochondria at physiological I, and offer further evidence that mitochondrial electron transport is a process driven by random collisions between its independently diffusing electron transferring, redox components.

The ionic strength of the intermembrane space of intact mitochondria is not affected by the pH or volume of the intermembrane space.

Ionic strength affects the electron transport activity of cytochrome c through its electrostatic interactions with redox partners and membrane lipids. We previously reported (Cortese, J.D., Voglino, A.L. and Hackenbrock, C.R. (1991) J. Cell Biol. 113, 1331-1340) that the ionic strength (I) of the intermembrane space (IMS-I) in isolated, intact condensed mitochondria is similar to the external, bulk I, over a wide range of bulk I. We now consider the possible effects of IMS-pH and IMS-volume, both variable parameters of mitochondrial function in situ, on IMS-I. IMS-pH and IMS-I were measured with pH- and I-sensitive fluorescent probes (highly fluorescent FITC-dextran for IMS-pH and FITC-BSA for IMS-I). These probes were delivered into the IMS of intact mitochondria via probe encapsulation into asolectin vesicles, followed by low pH-induced fusion of the vesicles with the outer membranes of intact mitochondria. IMS-pH was found to be 0.4-0.5 units lower than bulk pH over the pH range 6.0-8.5 for mitochondria with a large IMS-volume separating the two mitochondrial membranes (condensed configuration), and 0-0.2 units lower for mitochondria with a small IMS-volume and membranes closely opposed (orthodox configuration). This small pH difference between IMS-pM and bulk pH did not influence the similarity between IMS-I and bulk I. When the IMS-volume was osmotically decreased, bringing the two mitochondrial membranes in close proximity as in the orthodox configuration, IMS-I followed the bulk I above 10 mM but did not respond to changes in bulk I below 10 mM. The lack of response of the IMS-I below 10 mM indicates that the close proximity of the two mitochondrial membranes excludes ions only at low, nonphysiological I. Since the similarity of IMS-I and bulk I is unaffected by either IMS-pH or IMS-volume above a bulk I of 10 mM, at cytosolic physiological I (i.e., 100-150 mM) cytochrome c can be expected to be a free, three-dimensional diffusant in the IMS irrespective of the pH or volume of the IMS.

Two-dimensional diffusion of F1F0-ATP synthase and ADP/ATP translocator. Testing a hypothesis for ATP synthesis in the mitochondrial inner membrane.

We report here the first experimentally determined lateral diffusion coefficients of the F1F0-ATP synthase and the ADP/ATP translocator in isolated inner membranes of rat liver mitochondria. Rabbit IgG developed against the F1F0-ATP synthase isolated from rat liver mitochondria was determined to be immunospecific for the synthase subunits, notably the alpha-beta doublet, gamma and delta subunits of F1 and subunits two, three and four of F0. 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 bc1 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 F1F0-ATP synthase was determined to be 8.4 x 10(-10) cm2/s or twice that of cytochrome bc1 complex while the diffusion coefficient of the ADP/ATP translocator was 1.7 x 10(-9) cm2/s or four times that of cytochrome bc1 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 F1F0-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.

Dynamics, structure, and function are coupled in the mitochondrial matrix.

The coupling between molecular diffusion and the structure and function of the rat liver mitochondrial matrix was explored using fluorescence anisotropy techniques and electron microscopy. The results confirm that matrix ultrastructure and the concentration of matrix protein are influenced by the respiratory state of mitochondria and the osmolarity of the external medium. At physiological osmolarity, a fluorescent metabolite-sized probe was found to diffuse slowly in the mitochondrial matrix but not to be completely immobile. In addition, significant differences in diffusion rates were found to exist between different mitochondrial respiratory states, with the slowest diffusion occurring in states with the highest matrix protein concentration. These data support the concept of a matrix structure in which diffusion is considerably hindered due to limited probe-accessible water and further suggest that volume-dependent regulation of matrix protein packing may modulate metabolite diffusion and, in turn, mitochondrial metabolism.

The mobility of a fluorescent ubiquinone in model lipid membranes. Relevance to mitochondrial electron transport.

The diffusion and location of a functional, fluorescent ubiquinone molecule, NBDHA-Q, were determined as a function of temperature using microscopic observation, fluorescence recovery after photobleaching and fluorescence spectroscopy in protein-free, pure-lipid dimyristoylphosphatidylcholine and dimyristoylphosphatidylcholine/cholesterol multibilayers. The data reveal that in a liquid-crystalline membrane (1) ubiquinone is highly mobile, (2) ubiquinone uniformly diffuses laterally with the same diffusion coefficient (3.10(-8) cm2/s at 25 degrees C) as the phospholipids in which it resides, (3) the diffusion coefficients of ubiquinone and phospholipid both decrease at the exothermic phase transition of the phospholipid, (4) cholesterol affects the diffusion coefficients of ubiquinone and phospholipids to the same degree, (5) cholesterol induces a lateral phase separation progressively excluding ubiquinone from cholesterol-containing domains. These data suggest that ubiquinone does not reside at the membrane surface or in the mid-plane for any appreciable length of time. Rather, the data indicate that ubiquinone is highly mobile laterally and transversely, spending the majority of its time in the acyl chain region of the membrane, where its lateral and transverse diffusion is limited by the lateral diffusion and the transverse microviscosity gradient of the phospholipids and where its lateral location can be affected by the presence of cholesterol. In addition, based upon a comparison of the diffusion coefficients for ubiquinone, phospholipids and mitochondrial redox complexes, we hypothesize that no significant portion of the ubiquinone pool remains bound to redox complexes for any significant length of time relative to that for electron transport as resolvable by fluorescence recovery after photobleaching.

Ionic strength of the intermembrane space of intact mitochondria as estimated with fluorescein-BSA delivered by low pH fusion.

The electrostatic interactions of cytochrome c with its redox partners and membrane lipids, as well as other protein interactions and biochemical reactions, may be modulated by the ionic strength of the intermembrane space of the mitochondrion. FITC-BSA was used to determine the relative value of the mitochondrial intermembrane ionic strength with respect to bulk medium external to the mitochondrial outer membrane. FITC-BSA exhibited an ionic strength-dependent fluorescence change with an affinity in the mM range as opposed to its pH sensitivity in the microM range. A controlled, low pH-induced membrane fusion procedure was developed to transfer FITC-BSA encapsulated in asolectin liposomes, to the intermembrane space of intact mitochondria. The fusion procedure did not significantly affect mitochondrial ultrastructure, electron transport, or respiratory control ratios. The extent of fusion of liposomes with the mitochondrial outer membrane was monitored by fluorescence dequenching assays using a membrane fluorescent probe (octadecylrhodamine B) and the soluble FITC-BSA fluorescent probe, which report membrane and contents mixing, respectively. Assays were consistent with a rapid, low pH-induced vesicle-outer membrane fusion and delivery of FITC-BSA into the intermembrane space. Similar affinities for the ionic strength-dependent change in fluorescence were found for bulk medium, soluble (9.8 +/- 0.8 mM) and intermembrane space-entrapped FITC-BSA (10.2 +/- 0.6 mM). FITC-BSA consistently reported an ionic strength in the intermembrane space of the functionally and structurally intact mitochondria within +/- 20% of the external bulk solution. These findings reveal that the intermembrane ionic strength changes as does the external ionic strength and suggest that cytochrome c interactions, as well as other protein interactions and biochemical reactions, proceed in the intermembrane space of mitochondria in the intact cell at physiological ionic strength, i.e., 100-150 mM.

Lateral diffusion of redox components in the mitochondrial inner membrane is unaffected by inner membrane folding and matrix density.

We report the first lateral diffusion measurements of redox components in normal-sized, matrix-containing, intact mitoplasts (inner membrane-matrix particles). The diffusion measurements were obtained by submicron beam fluorescence recovery after photobleaching measurements of individual, intact, rat liver mitoplasts bathed in different osmolarity media to control the matrix density and the extent of inner membrane folding. The data reveal that neither the extent of mitochondrial matrix density nor the complexity of the inner membrane folding have a significant effect on the mobility of inner membrane redox components. Diffusion coefficients for Complex I (NADH:ubiquinone oxidoreductase), Complex III (ubiquinol: cytochrome c oxidoreductase), Complex IV (cytochrome oxidase), ubiquinone, and phospholipid were found to be effectively invariant with the matrix density and/or membrane folding and essentially the same as values we reported previously for spherical, fused, ultralarge, matrix-free, inner membranes. Diffusion of proton-transporting Complex V (ATP synthase) appeared to be 2-3-fold slower at the greatest matrix density and degree of membrane folding. Consistent with a diffusion-coupled mechanism of electron transport, comparison of electron transport frequencies (productive collisions) with the theoretical, diffusion-controlled, collision frequencies (maximum collisions possible) revealed that there were consistently more calculated than productive collisions for all redox partners. Theoretical analyses of parameters for submicron fluorescence recovery after photobleaching measurements in intact mitoplasts support the finding of highly mobile redox components diffusing at the same rates as determined in conventional fluorescence recovery after photobleaching measurements in fused, ultralarge inner membranes. These findings support the Random Collision Model of Mitochondrial Electron Transport at the level of the intact mitoplast and suggest a similar conclusion for the intact mitochondrion.

On the measurement of particle number and mobility in nonideal solutions by fluorescence correlation spectroscopy.

Interparticle interactions are incorporated into the theoretical description of the initial amplitude, G(0), of the normalized fluorescence correlation spectroscopy autocorrelation function. Measurements of particle number, aggregate size, and interaction-dependent diffusion are then analyzed in the context of this generalized theory. It is shown that the neglect of interactions can introduce order-of-magnitude errors into estimates of particle number and aggregate size. It is also shown that measurement of G(0) provides an essentially unique method for testing the validity of theories of interaction-dependent membrane protein diffusion.

Lateral diffusion as a rate-limiting step in ubiquinone-mediated mitochondrial electron transport.

Data are presented which indicate that the diffusion-based collisions of ubiquinone with its redox partners in the mitochondrial inner membrane are a rate-limiting step for maximum (uncoupled) rates of succinate-linked electron transport. Data were obtained from experimental analysis of a comparison of the apparent activation energies of lateral diffusion rates, collision frequencies, and electron transport rates in native and protein-diluted (phospholipid-enriched) inner membranes. Diffusion coefficients for Complex III (ubiquinol:cytochrome c oxidoreductase) and ubiquinone redox components were determined as a function of temperature using fluorescence recovery after photobleaching, and collision frequencies of appropriate redox partners were subsequently calculated. The data reveal that 1) the apparent activation energies for both diffusion and electron transport were highest in the native inner membrane and decreased with decreasing protein density, 2) the apparent activation energy for the diffusion step of ubiquinone made up the most significant portion of the activation energy for the overall kinetic activity, i.e. electron transport steps plus the diffusion steps, 3) the apparent activation energies for both diffusion and electron transport decreased in a proportionate manner as the membrane protein density was decreased, and 4) Arrhenius plots of the ratio of experimental electron transport productive collisions (turnovers) to calculated theoretically predicted, diffusion-based collisions for ubiquinone with its redox partners had little or no temperature dependence, indicating that as temperature increases, increases in electron transport rate are accounted for by the increases in diffusion-based collisions. These data support the Random Collision Model of mitochondrial electron transport in which the rates of diffusion and appropriate concentrations of redox components limit the maximum rates of electron transport in the inner membrane.

The multicollisional, obstructed, long-range diffusional nature of mitochondrial electron transport.

Data are presented which reveal that ubiquinone (Q)-mediated electron transport is a multicollisional, obstructed, long-range diffusion process, where factors that affect the rate of lateral diffusion also affect the rate of electron transport. Based on fluorescence recovery after photobleaching measurements, it was concluded that Q-mediated electron transport occurs by the random collision of redox components which are independent lateral diffusants, each greater than 86% mobile and diffusing in a common pool. The diffusion process of Q-mediated electron transport is 1) multicollisional since the transfers of reducing equivalents between appropriate redox partners occur with less than 100% collision efficiency; 2) obstructed since its maximal rate as well as the rates of diffusion of all redox components involved vary as a function of the membrane protein density; and 3) long-range since the diffusion of all redox components is protein density-dependent, and the diffusion distance required for Q to catalyze the transfer of a reducing equivalent from Complex II to III must be, on average, greater than 37.6 nm. These findings and other theoretical treatments reveal that measurements of short-range diffusion (less than 10 nm), in which collisions between appropriate redox partners do not occur, on average, and which are not affected by membrane protein density, are irrelevant to the collisional process of electron transport. Thus, the data show that the maximum electron transport rate is dependent on both the diffusion rate and the concentration of the redox components. Sucrose was found to inhibit both the mobility of redox components as well as their electron transport rates. Data presented on the relationships between membrane viscosity, rates of lateral and rotational diffusion, and mobile fractions of redox components do not support rotationally immobile aggregates in the functional inner membrane. The high degree of unsaturated phospholipids and the absence of cholesterol in the bilayer of the native inner membrane reflect a requirement for a low resistance to motion of the redox components to compensate for the multicollisional, obstructive nature of their catalytically important collisions in this membrane. These findings support the Random Collision Model of electron transport in which the diffusion and concentration of redox components limit the maximum rate of electron transport.

Multidimensional diffusion modes and collision frequencies of cytochrome c with its redox partners.

We have determined the modes and rates of cytochrome c diffusion as well as the collision frequencies of cytochrome c with its redox partners at the surface of the isolated, mitochondrial inner membrane over a broad range (0-150 mM) of ionic strengths. Using fluorescence recovery after photobleaching, resonance energy transfer, and direct binding assay, we determined that the diffusion coefficient of cytochrome c is independent of its concentration and quantity bound to the inner membrane, that the distance of cytochrome c from the membrane surface increases with increasing ionic strength, and that there is no significant immobile fraction of cytochrome c on the membrane regardless of ionic strength. The rate of cytochrome c diffusion increases while its mode of diffusion changes progressively from lateral to three-dimensional with increasing ionic strength. At physiological ionic strength (100-150 mM), the diffusion of cytochrome c is three-dimensional with respect to the surface of the inner membrane with a coefficient of 1.0 x 10(-6) cm2/s, and little, if any cytochrome c is bound to the membrane regardless of its concentration. Furthermore, as ionic strength is raised from zero to 150 mM, the cytochrome ckd for the inner membrane increases, its mean occupancy time on the inner membrane to collide with a redox partner (tau) decreases, and its diffusion-based collision frequencies with its redox partners decrease. These data reveal the significance of both diffusion and concentration (affinity) of cytochrome c near the surface of the inner membrane in the control of the collision frequency of cytochrome c with its redox partners.

The role of cytochrome c diffusion in mitochondrial electron transport.

We have compared the modes and rates of cytochrome c diffusion to the rates of cytochrome c-mediated electron transport in isolated inner membranes and in whole intact mitochondria. For inner membranes, an increasing ionic strength results in an increasing rate of cytochrome c diffusion, a decreasing concentration (affinity) of cytochrome c near the membrane surface as well as near its redox partners, and an increasing rate of electron transport. For intact mitochondria, an increasing ionic strength results in a parallel, increasing rate of cytochrome c-mediated electron transport. In both inner membranes and intact mitochondria the rate of cytochrome c-mediated electron transport is highest at physiological ionic strength (100-150 mM), where the diffusion rate of cytochrome c is highest and its diffusion mode is three-dimensional. In intact mitochondria, succinate and duroquinol-driven reduction of endogenous cytochrome c is greater than 95% at all ionic strengths, indicating that cytochrome c functions as a common pool irrespective of its diffusion mode. Using a new treatment to obtain bimolecular diffusion-controlled collision frequencies in a heterogenous diffusion system, where cytochrome c diffuses laterally, pseudo-laterally, or three-dimensionally while its redox partners diffuse laterally, we determined a high degree of collision efficiency (turnover/collisions) for cytochrome c with its redox partners for all diffusion modes of cytochrome c. At physiological ionic strength, the rapid diffusion of cytochrome c in three dimensions and its low concentration (affinity) near the surface of the inner membrane mediate the highest rate of electron transport through maximum collision efficiencies. These data reveal that the diffusion rate and concentration of cytochrome c near the surface of the inner membrane are rate-limiting for maximal (uncoupled) electron transport activity, approaching diffusion control.

The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport.

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.

Variation in protein lateral diffusion coefficients is related to variation in protein concentration found in mitochondrial inner membranes.

The electrophoretic freeze-fracture electron microscopy method (Sowers, A.E. and Hackenbrock, C.R. (1984) Proc. Natl. Acad. Sci. USA 78, 6246-6250) for measuring the lateral diffusion coefficient of integral proteins was applied to a large population of spherical-shaped mitochondrial inner membranes. Membrane integral protein concentration was estimated by determining the intramembrane particle concentration. Analysis of the data reveals that: (a) the radii of the spherical inner membranes in the selected population ranged from 0.22 to 1.2 micron, (b) the intramembrane particle concentrations ranged from 2300 to 6400 per micron2, and (c) the calculated lateral diffusion coefficients of the intramembrane particles ranged from 1.3 X 10(-10) to 3.35 X 10(-9) cm2/s. The data clearly show a naturally occurring large range in protein concentration in the mitochondrial inner membrane and an inverse correlation of lateral diffusion coefficient with the membrane protein concentration. This study is the first to show that the lateral diffusion coefficient of integral proteins in a native membrane varies as the membrane protein concentration.

Calcium-mediated fusion to produce ultra large osmotically active mitochondrial inner membranes of controlled protein density.

We have developed a new membrane fusion method which produces ultra large, spherical mitochondrial inner membranes attached to microscope slides. The fused inner membranes measured up to 200 microns in diameter. The technique fuses native inner membranes as well as inner membranes in which the protein density has been varied by enriching with exogenous phospholipid. The fusion process is accomplished through the use of calcium, low pH and elevated temperature. Characterization of the fused membranes was carried out using phase, fluorescence, and freeze-fracture electron microscopy. These ultra large, fused inner membranes were found to model the inner membranes from which they were formed. The fused inner membranes were found to be osmotically active and are large enough for measuring the lateral diffusion of membrane components by fluorescence recovery after photobleaching and are large enough for microelectrode impalement.

Independent lateral diffusion of cytochrome bc1 complex and cytochrome oxidase in the mitochondrial inner membrane.

Distinct fluorophores have been conjugated to antibodies for cytochrome bc1 complex and cytochrome oxidase, two integral electron transferring proteins in the mitochondrial inner membrane. Addition of these fluorescent antibodies to preparations of mitochondrial inner membranes followed by appropriate secondary antibodies causes distinct and independent aggregation of the two cytochrome proteins. These results reveal that both cytochrome bc1 complex and cytochrome oxidase diffuse laterally in the membrane plane independent of one another consistent with the random collision model for electron transport in the mitochondrial inner membrane.