Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Reactivity with ubiquinone of quinoprotein D-glucose dehydrogenase from Gluconobacter suboxydans

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent oxidoreductase linked to the respiratory chain of a wide variety of bacteria. There is a controversy as to whether the glucose dehydrogenase is linked to the respiratory chain via ubiquinone or cytochrome b. In this study, it was shown that the glucose dehydrogenase of Gluconobacter suboxydans has the ability to react directly with ubiquinone. The enzyme purified from the membranes of G. suboxydans was able to react with ubiquinone homologues such as ubiquinone-1, -2, or -6 in detergent solution. Furthermore, in order to demonstrate the reactivity of the enzyme with native ubiquinone, ubiquinone-10, in the native membranous environment, the dehydrogenase was reconstituted together with cytochrome o, the terminal oxidase of the respiratory chain, into a phospholipid bilayer containing ubiquinone-10. The proteoliposomes thus reconstituted exhibited a reasonable glucose oxidase activity, the electron transfer reaction of which was able to generate a membrane potential and a pH gradient. Thus, D-glucose dehydrogenase of G. suboxydans has been demonstrated to donate electrons directly to ubiquinone in the respiratory chain.     

Evidence for electron transfer via ubiquinone between quinoproteins D-glucose dehydrogenase and alcohol dehydrogenase of Gluconobacter suboxydans

Gluconobacter suboxydans contains membrane-bound D-glucose and alcohol dehydrogenases (GDH and ADH) as the primary dehydrogenases in the respiratory chain. These enzymes are known to be quinoproteins having pyrroloquinoline quinone as the prosthetic group. GDH reduces an artificial electron acceptor, ferricyanide, in the membrane, but not after solubilization with Triton X-100, while ADH can react with the electron acceptor even after solubilization and further purification. In this study, it has been shown that the ferricyanide reductase activity of GDH is restored by adding the supernatant solubilized with Triton X-100 to the residue, and also by incorporation of purified ADH into the membranes of an ADH-deficient strain. G. suboxydans var. alpha. In addition, the ferricyanide reductase activity of GDH was reconstituted in proteoliposomes from GDH, ADH, and ubiquinone-10. Thus, the results indicated that the electron transfer from GDH to ferricyanide was mediated by ubiquinone and ADH. The data also suggest that GDH and ADH transfer electrons mutually via ubiquinone in the respiratory chain.     

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. II. Direct electron transfer between the reaction center and the bc1 complex and role of cytochrome c2

1. The cyclic photosynthetic chain of Rhodobacter capsulatus has been reconstituted incorporating into phospholipid liposomes containing ubiquinone-10 two multiprotein complexes: the reaction center and the ubiquinol-cytochrome-c2 reductase (or bc1 complex). 2. In the presence of cytochrome c2 added externally, at concentrations in the range 10-10(4) nM, a flash-induced cyclic electron transfer can be observed. In the presence of antimycin, an inhibitor of the quinone-reducing site of the bc1 complex, the reduction of cytochrome b561 is a consequence of the donation of electrons to the photo-oxidized reaction center. At low ionic strength (10 mM KCl) and at concentrations of cytochrome c2 lower than 1 microM, the rate of this reaction is limited by the concentration of cytochrome c2. At higher concentrations the reduction rate of cytochrome b561 is controlled by the concentration of quinol in the membrane, and, therefore, is increased when the ubiquinone pool is progressively reduced. At saturating concentrations of cytochrome c2 and optimal redox poise, the half-time for cytochrome b561 reduction is about 3 ms. 3. At high ionic stength (200 mM KCl), tenfold higher concentrations of cytochrome c2 are required for promoting equivalent rates of cytochrome-b561 reduction. If the absolute values of these rates are compared with those of the cytochrome-c2-reaction-center electron transfer, it can be concluded that the reaction of oxidized cytochrome c2 with the bc1 complex is rate-limiting and involves electrstatic interactions. 4. A significant rate of intercomplex electron transfer can be observed also in the absence of cytochrome c2; in this case the electron donor to the recation center is the cytochrome c1 of the oxidoreductase complex. The oxidation of cytochrome c1 triggers a normal electron transfer within the bc1 complex. The intercomplex reaction follows second-order kinetics and is slowed at high ionic strength, suggesting a collisional interaction facilitated by electrostatic attraction. From the second-order rate constant of this process, a minimal bidimensional diffusion coefficient for the complexes in the membrane equal to 3 X 10(-11) cm2 s-1 can be evaluated.     

The indispensability of phospholipid and ubiquinone in mitochondrial electron transfer from succinate to cytochrome c.

The indispensability of phospholipid and ubiquinone (Q) in mitochondrial electron transfer was studied by depleting phospholipid and Q in succinate-cytochrome c reductase and then replenishing the depleted enzyme. More than 90% of phospholipid and Q was removed by repeated ammonium sulfate-cholate fractionation. The depleted succinate-cytochrome c reductase showed no enzymatic activity for succinate leads to c or QH2 leads to c and yet retained most of the succinate leads to Q activity. All enzymatic activity was restored upon the addition of Q and phospholipid. Restoration required the addition of Q prior to the addition of phospholipid. Reversing the addition sequence or addition of a mixture of phospholipid and Q resulted only in a small restoration of activities. The conditions for restoration are given in detail. Removal of phospholipid from succinate-cytochrome c reductase resulted in reduction of cytochrome c1 in the absence of exogenous electron donor. Replenishing the preparation with phospholipid brought about the reoxidation of cytochrome c1 in the absence of electron acceptor or oxygen.     

Effect of the nonionic detergent Triton X-100 on mitochondrial succinate-oxidizing enzymes

Specific activities of succinate:coenzyme Q reductase, ubiquinone:cytochrome c reductase, cytochrome oxidase, succinate:cytochrome c reductase, succinate oxidase, and ubiquinol oxidase have been measured in rat liver mitochondria in the presence of Triton X-100. The last three activities are much more sensitive to Triton X-100 than the first ones; the data suggest that the electron transport chain components cannot react with each other in the presence of the detergent. At least in the case of succinate:cytochrome c reductase, reconstitution of the detergent-treated membranes with externally added phospholipids reverses the inhibition produced by Triton X-100. These results support the idea that the respiratory chain components diffuse at random in the plane of the inner mitochondrial membrane; the main effect of the detergent would be to impair lateral diffusion by decreasing the area of lipid bilayer. When detergent-treated mitochondrial suspensions are centrifuged in order to separate the solubilized from the particulate material, only the first three enzyme activities mentioned above are found in the supernatants. After centrifugation, a latent ubiquinol:cytochrome c oxidase activity becomes apparent, whereas the same centrifugation process produces inhibition of cytochrome c oxidase in the presence of certain Triton X-100 concentrations. These effects could be due either to a selective solubilization of regulatory or catalytic subunits or to a conformational change of the enzyme-detergent complex.     

Is ubiquinone diffusion rate-limiting for electron transfer?

The different possible dispositions of the electron transfer components in electron transfer chains are discussed: (a) random distribution of complexes and ubiquinone with diffusion-controlled collisions of ubiquinone with the complexes, (b) random distribution as above, but with ubiquinone diffusion not rate-limiting, (c) diffusion and collision of protein complexes carrying bound ubiquinone, and (d) solid-state assembly. Discrimination among these possibilities requires knowledge of the mobility of the electron transfer chain components. The collisional frequency of ubiquinone-10 with the fluorescent probe 12-(9-anthroyl)stearate, investigated by fluorescence quenching, is 2.3 × 10⁹ M^–1 sec^–1 corresponding to a diffusion coefficient in the range of 10^–6 cm²/sec (Fato, R., Battino, M., Degli Esposti, M., Parenti Castelli, G., and Lenaz, G.,Biochemistry,25, 3378–3390, 1986); the long-range diffusion of a short-chain polar Q derivative measured by fluorescence photobleaching recovery (FRAP) (Gupte, S., Wu, E. S., Höchli, L., Höchli, M., Jacobson, K., Sowers, A. E., and Hackenbrock, C. R.,Proc. Natl. Acad. Sci. USA 81, 2606–2610, 1984) is 3×10^–9 cm²/sec. The discrepancy between these results is carefully scrutinized, and is mainly ascribed to the differences in diffusion ranges measured by the two techniques; it is proposed that short-range diffusion, measured by fluorescence quenching, is more meaningful for electron transfer than long-range diffusion measured by FRAP, or microcollisions, which are not sensed by either method. Calculation of the distances traveled by random walk of ubiquinone in the membrane allows a large excess of collisions per turnover of the respiratory chain. Moreover, the second-order rate constants of NADH-ubiquinone reductase and ubiquinol-cytochromec reductase are at least three orders of magnitude lower than the second-order collisional constant calculated from the diffusion of ubiquinone. The activation energies of either the above activities or integrated electron transfer (NADH-cytochromec reductase) are well above that for diffusion (found to be ca. 1 kcal/mol). Cholesterol incorporation in liposomes, increasing bilayer viscosity, lowers the diffusion coefficients of ubiquinone but not ubiquinol-cytochromec reductase or succinate-cytochromec reductase activities. The decrease of activity by ubiquinone dilution in the membrane is explained by its concentration falling below theK _m of the partner enzymes. It is calculated that ubiquinone diffusion is not rate-limiting, favoring a random model of the respiratory chain organization. It is not possible, however, to exclude solid-state assemblies if the rate of dissociation and association of ubiquinone is faster than the turnover of electron transfer.     

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.     

The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Restoration of ubiquinone-pool behaviour.

1. In the inner mitochondrial membrane, dehydrogenases and cytochromes appear to act independently of each other, and electron transport has been proposed to occur through a mobile pool of ubiquinone-10 molecules [Kröger & Klingenberg (1973) Eur. J. Biochem. 34, 358--368]. 2. Such behaviour can be restored to the interaction between purified Complex I and Complex III by addition of phospholipid and ubiquinone-10 to a concentrated mixture of the Complexes before dilution. 3. A model is proposed for the interaction of Complex I with Complex III in the natural membrane that emphasizes relative mobility of the Complexes rather than ubiquinone-10. Electron transfer occurs only through stoicheiometric Complex I-Complex III units, which, however, are formed and re-formed at rates higher than the rate of electron transfer.     

Diffusional effects in the steady state kinetics of ubiquinol cytochrome c reductase in bovine heart submitochondrial particles

The steady-state kinetics of ubiquinol cytochrome c reductase was investigated in submitochondrial particles using ubiquinol-1 as electron donor in media of increasing viscosities obtained by water-polyethylene glycol mixtures. The minimum association rate constant, kmin = kcat/km, for cytochrome c was strongly viscosity dependent, whereas kmin for ubiquinol-1 was only weakly affected by viscosity. It is concluded that the interaction of cytochrome c with the membranous reductase is largely under diffusion control, whereas the oxidation of ubiquinol by the enzyme is not significantly controlled by diffusion in either the aqueous medium or the membrane. The results are compatible with the presence of a diffusion limited step in cytochrome c but not in ubiquinone in mitochondrial electron transfer.     

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 oxidation of external NADH by an intermembrane electron transfer in mitochondria from the ubiquinone-deficient mutant E3-24 of Saccharomyces cerevisiae

Cells of the E3-24 mutant of the strain D273-10B of Saccharomyces cerevisiae, grown in a fermentable substrate not showing catabolite repression of respiration (2% galactose), are able to respire, in spite of their ubiquinone deficiency in mitochondrial membranes. Mitochondria isolated from these mutant cells oxidize exogenous NADH through a pathway insensitive to antimycin A but inhibited by cyanide. Addition of methanolic solutions of ubiquinone homologs stimulates the oxidation rate and restores antimycin A sensitivity in both isolated mitochondria and whole cells. Mersalyl preincubation of isolated mitochondria inhibits both NADH oxidation and NADH-cytochrome c oxido-reductase activity (assayed in the presence of cyanide) with the same pattern. Electrons resulting from the oxidation of exogenous NADH reduce both cytochrome b5 and endogenous cytochrome c. The increase in ionic strength stimulates NADH oxidation, which is also coupled to the ATP synthesis with an ATP/O ratio similar to that obtained with ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) as substrate. The effect of cyanide on these activities and on NADH-induced endogenous cytochrome c reduction is also comparable. These results support the existence in vivo and in isolated mitochondria of a energy-conserving pathway for the oxidation of cytoplasmatic NADH not related to the dehydrogenases of the inner membrane, the ubiquinone, and the b-c1 complex, but involving a cytochrome c shuttle between the NADH-cytochrome c reductase of the outer membrane and cytochrome oxidase in the inner membrane.     

Succinate-ubiquinone reductase linked recycling of alpha-tocopherol in reconstituted systems and mitochondria: requirement for reduced ubiquinone.

Studies have demonstrated that accumulation of mitochondrial tocopheroxyl radical, the primary oxidation product of alpha-tocopherol, accompanies rapid consumption of tocopherol. Enzyme-linked electron flow lowers both the steady-state concentration of the radical and the consumption of tocopherol. Reduction of tocopheroxyl radical by a mitochondrial electron carrier(s) seems a likely mechanism of tocopherol recycling. Succinate-ubiquinone reductase (complex II) was incorporated into liposomes in the presence of tocopherol and ubiquinone-10. After inducing formation of tocopheroxyl radical, it was possible to show that reduced ubiquinone prevents radical accumulation and tocopherol consumption. There was no evidence of direct reduction of tocopheroxyl radical by succinate-reduced complex II. These reactions were also measured using ubiquinone-1 and alpha-C-6-chromanol (2,5,7,8-tetramethyl-2-(4'-methylpentyl)-6-chromanol) which are less hydrophobic analogues of ubiquinone-10 and alpha-tocopherol. Mitochondrial membranes were made deficient in ubiquinone but sufficient in alpha-tocopherol and were reconstituted with added quinone. With these membranes it was shown that mitochondrial enzyme-linked reduction of ubiquinone protects alpha-tocopherol from consumption, and there is a requirement for ubiquinone. This complements the observations made in liposomes and we propose that reduced mitochondrial ubiquinones have a role in alpha-tocopherol protection, presumably through efficient reduction of the tocopheroxyl radical.     

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers: the response under conditions of multiple reaction-center turnovers

The characteristics of the photocurrent response activated by continuous illumination of planar bilayer membranes containing bacterial reaction centers have been resolved by voltage clamp methods. The photocurrent response to a long light pulse consists of an initial spike arising from the fast, quasi-synchronous electron transfer from the reaction center bacteriochlorophyll dimer, BChl2, to the primary quinone QA. This is followed by a slow relaxation of the current to that promoted by secondary, asynchronous multiple electron transfers from the reduced cytochrome c through the reaction centers to the ubiquinone-10 pool. Currents derived from cytochrome c oxidation that occurs when cytochrome c is associated with the reaction center or when limited by diffusional interaction from solution are recognized. Changes of the ionic strength and pH in the aqueous phase, and the clamped membrane potential (+/- 150 mV), affect the electron-transfer rate between cytochrome c and BChl2. In contrast, the primary light-induced charge separation between BChl2 and QA, or electron transfer between QA on the ubiquinone pool are unaffected. During illumination of reaction center membranes supplemented with cytochrome c and a ubiquinone pool, there is a small but significant steady-state current which is considered to be caused by the re-oxidation of photoreduced quinone by molecular oxygen. In the dark, after illumination of reaction centers supplemented with cytochrome c and a ubiquinone pool, there is a small amount of reverse current resulting from the movement of charges back across the membrane. This reverse current is observed maximally after 400 ms illumination while prolonged illumination diminishes the effect. The source of this current is uncertain, but it is considered to be due to the flux of anionic semiquinone within the membrane profile; this may also be the species that interacts with oxygen giving rise to the steady-state current. It is postulated that when the reaction centers are contained in an alkane-containing phospholipid membrane, in contrast to the in vivo situation, the semiquinone anion formed in the QB site is not tightly bound to the site and can, by exchange-diffusion with the membrane-quinone pool, move away from the site and accumulate in the membrane. However, in the absence, more quantitative work superoxide anion, resulting from O2 interaction with semiquinone of QA, QB or pool cannot be excluded.     

Ethanol Oxidase Respiratory Chain of Acetic Acid Bacteria. Reactivity with Ubiquinone of Pyrroloquinoline Quinone-dependent Alcohol Dehydrogenases Purified from Acetobacter aceti and Gluconohacter suhoxydans

Membrane-bound, pyrroloquinoline quinone-dependent, alcohol dehydrogenase functions as the primary dehydrogenase in the respiratory chain of acetic acid bacteria. In this study, an ability of the enzyme to directly react with ubiquinone was investigated in alcohol dehydrogenases purified from both Acetobacter aceti and Gluconobacter suboxydans by two different approaches. First, it was shown that the enzymes are able to reduce natural ubiquinones, ubiquinone-9 or -t0, in a detergent solution as well as a soluble short-chain homologue, ubiquinone-I. In order to show the reactivity of the enzyme with natural ubiquinone in a native membrane environment, furthermore, alcohol dehydrogenase was reconstituted into proteoliposomes together with natural ubiquinone and a terminal ubiquinol oxidase. The reconstitution was done by binding the detergent-free dehydrogenase at room temperature to proteoliposomes that had been prepared in advance from a ubiquinol oxidase and phospholipids containing ubiquinone by detergent dialysis using octyl-β-D-glucopyranoside; the enzyme of A. aceti was reconstituted together with ubiquinone-9 and A. aceti cytochrome a₁ while G. suboxydans alcohol dehydrogenase was done into proteoliposomes containing ubiquinone-10 and G. suboxydans cytochrome o. The proteoliposomes thus reconstituted had a reasonable level of ethanol oxidase activity, the electron transfer reaction of which was also able to generate a ‘membrane potential. Thus, it has been shown that alcohol dehydrogenase of acetic acid bacteria donates electrons directly to ubiquinone in the cytoplasmic membranes and thus the ethanol oxidase respiratory chain of acetic acid bacteria is constituted of only three membranous respiratory components, alcohol dehydrogenase, ubiquinone, and terminal ubiquinol oxidase.     

Mobility in the mitochondrial electron transport chain

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.     

Determination of partition and lateral diffusion coefficients of ubiquinones by fluorescence quenching of n-(9-anthroyloxy)stearic acids in phospholipid vesicles and mitochondrial membranes

The quenching of fluorescence of n-(9-anthroyloxy)stearic acids and other probes by different ubiquinone homologues and analogues has been exploited to assess the localization and lateral mobility of the quinones in lipid bilayers of model and mitochondrial membranes. The true bimolecular collisional quenching constants in the lipids together with the lipid/water partition coefficients were obtained from Stern-Volmer plots at different membrane concentrations. A monomeric localization of the quinone in the phospholipid bilayer is suggested for the short side-chain ubiquinone homologues and for the longer derivatives when cosonicated with the phospholipids. The diffusion coefficients of the ubiquinones, calculated from the quenching constants either in three dimensions or in two dimensions, are in the range of (1-6) X 10(-6) cm2 s-1, both in phospholipid vesicles and in mitochondrial membranes. A careful analysis of different possible locations of ubiquinones in the phospholipid bilayer, accounting for the calculated diffusion coefficients and the viscosities derived therefrom, strongly suggests that the ubiquinone 10 molecule is located within the lipid bilayer with the quinone ring preferentially adjacent to the polar head groups of the phospholipids and the hydrophobic tail largely accommodated in the bilayer midplane. The steady-state rates of either ubiquinol 1-cytochrome c reductase or NADH:ubiquinone 1 reductase are proportional to the concentration of the quinol or quinone substrate in the membrane. The second-order rate constants appear to be at least 3 orders of magnitude lower than the second-order constants for quenching of the fluorescent probes; this is taken as a clear indication that ubiquinone diffusion is not the rate-determining step in the quinone-enzyme interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of a reconstitutively active iron-sulfur protein (oxidation factor) from succinate . cytochrome c reductase complex of bovine heart mitochondria.

Oxidation factor, a protein required for electron transfer from succinate to cytochrome c in the mitochondrial respiratory chain, has been purified from isolated succinate . cytochrome c reductase complex. Purification of the protein has been followed by a reconstitution assay in which restoration of ubiquinol . cytochrome c reductase activity is proportional to the amount of oxidation factor added back to depleted reductase complex. The purified protein is a homogeneous polypeptide on acrylamide gel electrophoresis in sodium dodecyl sulfate and migrates with an apparent Mr = 24,500. Purified oxidation factor restores succinate . cytochrome c reductase and ubiquinol . cytochrome c reductase activities to depleted reductase complex. It is not required for succinate dehydrogenase nor for succinate . ubiquinone reductase activities of the reconstituted reductase complex. Oxidation factor co-electrophoreses with the iron-sulfur protein polypeptide of ubiquinol . cytochrome c reductase complex. The purified protein contains 56 nmol of nonheme iron and 36 nmol of acid-labile sulfide/mg of protein and possesses an EPR spectrum with the characteristic "g = 1.90" signal identical to that of the iron-sulfur protein of the cytochrome b . c1 complex. In addition, the optimal conditions for extraction of oxidation factor, including reduction with hydrosulfite and treatment of the b . c1 complex with antimycin, are identical to those which facilitate extraction of the iron-sulfur protein from the b . c1 complex. These results indicate that oxidation factor is a reconstitutively active form of the iron-sulfur protein of the cytochrome b . c1 complex first discovered by Rieske and co-workers (Rieske, J.S., Maclennan, D.H., and Coleman, R. (1964) Biochem. Biophys. Res. Commun. 15, 338-344) and thus demonstrate that this iron-sulfur protein is required for electron transfer from ubiquinol to cytochrome c in the mitochondrial respiratory chain.     

Studies On The Role Of Ubiquinone In The Control Of The Mitochondrial Respiratory Chain

This study examines the possible role of Coenzyme Q (CoQ. ubiquinone) in the control of mitochondrial electron transfer. The CoQ concentration in mitochondria from different tissues was investigated by HPLC. By analyzing the rates of electron transfer as a function of total CoQ concentration, it was calculated that, at physiological CoQ concentration NADH cytochrome c reductase activity is not saturated. Values for theoretical V_max could not be reached experimentally for NADH oxidation, because of the limited mis-cibility of CoQ₁₀ with the phospholipids. On the other hand, it was found that CoQ₃ could stimulate α-glycerophosphate cytochrome c reductase over three-fold. Electron transfer being a diffusion-coupled process. we have investigated the possibility of its being subjected to diffusion control. A reconstruction study of Complex I and Complex III in liposomes showed that NADH cytochrome c reductase was not affected by changing the average distance between complexes by varying the protein: lipid ratios. The results of a broad investigation on ubiquinol cytochrome c reductase in bovine heart submitochondrial particles indicated that the enzymic rate is not diffusion-controlled by ubiquinol. whereas the interaction of cytochrome c with the enzyme is clearly diffusion-limited     

The interaction of NADH-cytochrome b5 reductase and cytochrome b5 bound to egg lecithin liposomes.

Incubation of liposomes prepared by sonication of egg lecithin with the amphipathic form of cytochrome b5 results in the binding of a maximum of 244 molecules of cytochrome b5 per liposomal vesicle. Interactions of the phospholipid with the hydrophobic segment of cytochrome b5 are involved in this binding which does not disrupt the liposome. When a small amount of NADH-cytochrome b5 reductase is bound liposomes simultaneously with cytochrome b5, the two proteins catalyze the reduction of cytochrome c by NADH. A qualitative kinetic analysis reveals that all of the cytochrome b5 interacts with reductase, a result consistent with these protein undergoing translational diffusion in the plane of the membrane. This system and the purified stearyl coenzyme A desaturase provide a model to study the dynamics of protein andlipid interactions in this membrane-bound oxidative sequence.