Inhibition of electron transfer through the cytochrome b-c 1 complex by nitric oxide in a photodenitrifier,Rhodopseudomonas sphaeroides forma sp. denitrificans

The effects of nitric oxide (NO) on electron transfer were studied with a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans. NO inhibited the oxidation of cytochrome c induced by continuous illumination in intact cells. NO inhibited the re-reduction of cytochrome c, the slow phase of the carotenoid bandshift, and the oxidation of cytochrome b after a flash illumination, suggesting that NO inhibited the photosynthetic cyclic electron transfer through the cytochrome b-c ₁ region. NO also inhibited the nitrite (NO ₂ ^- ) and NO reductions with succinate as the electron donor in intact cells, but did not inhibit the NO ₂ ^- and NO reductions in chromatophore membranes with ascorbate and phenazine methosulfate as the electron donors. NO reversibly inhibited the ubiquinol: cytochrome c oxidoreductase of the membranes, suggesting that NO inhibited the electron transfer through the cytochrome b-c ₁ region and that the cytochrome b-c ₁ complex also was involved in the electron transport in both NO ₂ ^- and NO reductions. The catalytic site of NO reduction was distinct from the inhibitory site of NO.Abbreviations UHDBT 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole - UHNQ 3-undecyl-2-hydroxy-1,4-naphthoquinone - MOPS 3-(N-morpholino)propane-sulfonic acid - PMS phenazine methosulfate - DCIP 2,6-dichlorophenol indophenol - DDC diethyl-dithiocarbamate     

The nature of the inhibition of 4,7-dioxobenzothiazole derivatives on mitochondrial ubiquinol-cytochrome c reductase

To investigate the inhibitory action and binding site of a quinone-like molecule, 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), a series of 4,7-dioxobenzothiazole derivatives were synthesized and their inhibitory efficiencies studied. Replacing the 6-hydroxyl or 2-hydrogen of UHDBT with a bromo or a methoxy group causes only a slight decrease in inhibitory efficiency, indicating that the 6-hydroxyl or the 2-hydrogen of UHDBT is not a structural requirement for inhibition. 5-Undecyl-6-bromo (or methoxy)-4,7-dioxobenzothiazole shows a pH-dependent inhibition similar to that observed with UHDBT, suggesting that the pH dependence is due to the presence of a dissociable group in the protein complex and not to the deprotonation of the hydroxyl group of the inhibitor. Replacing the 6-hydroxyl group with an azido group causes changes similar to those observed with UHDBT; the inhibition is accompanied by alteration of the epr characteristics of reduced iron-sulfur protein in ubiquinol-cytochrome c reductase. The extent of inhibition is not changed upon illumination of the treated reductase. When the photolyzed, 6-azido-5-(1',2'-[3H] undecyl)-4,7-dioxobenzothiazole [( 3H]6-azido-UDBT)-treated reductase is subjected to organic solvent extraction, no radioactivity is found in the reductase protein. Rather, the radioactivity is located in the phospholipid fraction. A [3H]azido-UDBT-cardiolipin adduct, identified after separation of the phospholipid fraction by high performance liquid chromatography, has 6-azido-UDBT linked to an acyl group, not to the head group of the cardiolipin molecule. These results suggest that inhibition by UHDBT is due to perturbation of specific cardiolipin molecules in ubiquinol-cytochrome c reductase. Since UHDBT and 6-azido-UDBT also inhibit the ubiquinol-cytochrome c reductase activity of delipidated reductase (10% of the original lipid remaining) assayed after reconstitution with ubiquinone and phospholipid, and the [3H]azido-UDBT-cardiolipin adduct is also found in the delipidated reductase, the UHDBT-perturbed cardiolipin molecule is structurally indispensable to reductase and it tightly bound to the reductase protein, most likely the quinone binding proteins.     

Effects of myxothiazol and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole on the respiratory pathways of the phytopathogenic fluorescent bacteria Pseudomonas cichorii and Pseudomonas aptata

Myxothiazol inhibited the electron transport in the cytochrome b/c segment of membrane particles from Pseudomonas cichorii. A residual NADH-oxidation due to the presence of an alternative pathway via cytochrome o (Em,7=+250 mV) was sensitive to the quinone analog 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). This latter inhibitor was equally effective in blocking the linear respiratory chain of Pseudomonas aptata, a strain deficient in cytochromes of c type and Rieske iron-sulphur centre. The analysis of the oxido-reduction kinetic patterns of cytochromes indicated that, among the b type haems present in P. aptata, only cyt. o could be reduced by ubiquinol-1 in a reaction insensitive to both antimycin A and myxothiazol but inhibited by UHDBT. This latter finding has been correlated to the fact that P. aptata exhibits a defective b/c complex. In membranes from P. cichorii, in which the absorption maximum of dithionite reduced cytochrome(s) b shifted by 2–3 nm in the presence of antimycin A and/or myxothiazol, the electron flow through the b/c oxidoreductase complex has tentatively been arranged in a proton motive Q-cycle like mechanism.Non standard abbreviations UHDBT 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole - cyt. cytochrom - Em, 7 mid-point potential at pH 7.0 - b/c complex ubiquinol-cyt. c oxidoreductase     

Evidence that energy conserving electron transport pathways to nitrate and cytochrome o branch at ubiquinone in Paracoccus denitrificans

The organisation and function of electron transport pathways in Paracoccus denitrificans has been studied with both inhibitors and electrode probes for O₂ or N₂O respiration and membrane potential. Myxothiazol completely inhibits electron flow through the cytochrome bc₁ region of the electron transport chain, as judged by its effect on nitrous oxide respiration. Electron flow to oxygen via the cytochrome o oxidase was shown to be insensitive to myxothiazol in a mutant, HUUG 25, that lacks cytochrome c and in which the aa₃ oxidase is therefore inactive. Myxothiazol did not inhibit nitrate reduction. It is concluded that myxothiazol is a specific inhibitor of electron flow through the cytochrome bc₁ region and more potent than antimycin which does not give complete inhibition.As neither antimycin nor myxothiazol, nor a combination of the two, inhibits electron transport to either nitrate reductase or cytochrome o it is concluded that electron transport pathways to these enzymes do not involve the cytochrome bc₁ region but rather branch at the level of ubiquinone. Although the cytochrome o pathway branches at ubiquinone, it is associated with the generation of a protonmotive force; this is shown by measurements of membrane potential in vesicle preparations from the mutant HUUG 25.In contrast to antimycin and myxothiazol, the ubiquinone analogues 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-undecyl-3-hydroxy-1,4-naphthoquinone (UHNQ) inhibit electron flow through both the cytochrome bc₁ complex and the cytochrome o pathway, although a higher titre is required in the latter case. These two inhibitors were without effect on the nitrate reductase pathway. Thus myxothiazol is the inhibitor of choice for selective and complete inhibition of cytochrome bc₁.Recently published schemes for electron transport in P. denitrificans are analysed.Non standard abbreviations S-13 2,5-dichloro-3-t-butyl-4-nitrososalicylanilide - UHNQ 2-n-undecyl-3-hydroxy-1,4-naphthoquinone - UHDBT 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole     

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.     

Sulfide-quinone and sulfide-cytochrome reduction in Rhodobacter capsulatus

The reduction by sulfide of exogenous ubiquinone is compared to the reduction of cytochromes in chromatophores of Rhodobacter capsulatus. From titrations with sulfide values for V_max of 300 and 10 moles reduced/mg bacteriochlorophyll a·h, and for K_m of 5 and 3 M were estimated, for decyl-ubiquinone-and cytochrome c-reduction, respectively. Both reactions are sensitive to KCN, as has been found for sulfide-quinone reductase (SQR) in Oscillatoria limnetica, which is a flavoprotein. Effects of inhibitors interfering with quinone binding sites suggest that at least part of the electron transport from sulfide in R. capsulatus employs the cytochrome bc ₁-complex via the ubiquinone pool.Abbreviations BChl a bacteriochlorophyll a - DAD diaminodurene - decyl-UQ decyl-ubiquinone - LED light emitting diode - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - PQ-1 plastoquinone 1 - SQR sulfide-quinone reductase (E.C. 1.8.5.'.) - UQ ubiquinone 10 - Q_c the quinone reduction site on the cytochrome b ₆ f/bc ₁, complex (also termed Q_i or Q_r or Q_n) - Q_s the quinone reduction site on SQR - Q_z quinol oxidation site on the b ₆ f/bc ₁, complex (also termed Q_o or Q_p)     

Kinetic studies of the lipid requirement of mitochondrial cytochromec oxidase

The lipid requirement of cytochromec oxidase was reinvestigated using both acetone and phospholipase A to deplete mitochondria of lipid. Removal of lipid resulted in a decrease in both the apparentK _m for cytochromec and apparentV _max when compared to control mitochondria. Addition of phospholipid to the assay mixture reactivated the enzyme. For both treatments theK _m returned to the control value. With phospholipase A treated mitochondria theV _max increased to near the control value, while acetone extracted mitochondria could be restored to aV _max of 1/2 that of the control. Detergent does not substitute for phospholipid and inhibits the reactivation with phospholipid.This research was supported in part by United States Public Health Service Research Grant AM-14632 and a Grant-in-Aid of the American Heart Association.     

Inhibitory effect of α-tocopherol and its derivatives on bovine heart succinate-cytochrome c reductase

α-Tocopherol and its derivatives inhibit succinate-cytochrome c reductase activity at a concentration of 0.5 μmol/mg protein in 50 mM phosphate buffer, pH 7.4, containing 0.4 % sodium cholate when α-tocopherol is predispersed in sodium cholate solution. The inhibitory site is located at the cytochrome b-c₁ region. Succinate-ubiquinone reductase activity of succinate-cytochrome c reductase was not impaired by treatment with α-tocopherol. The α-tocopherol-inhibited succinate-cytochrome c reductase activity can be reversed by the addition of ubiquinone and its analogs. When ubiquinone- and phospholipid-depleted succinate-cytochrome c reductase was treated with α-tocopherol followed by reaction with a fixed amount of 2,3-dimethoxy-6-methyl-5-(10-bromodecyl)-1,4-benzoquinone and phospholipid, the amount of α-tocopherol needed to express the maximal inhibition was only 0.3 μmol/mg protein. When ubiquinone- and phospholipid-depleted enzyme was treated with a given amount of α-tocopherol and followed by titration with 2,3-dimethoxy-6-methyl-5-(10-bromodecyl)-1,4-benzoquinone, restoration of activity was enhanced at low concentrations of ubiquinone analog, indicating that α-tocopherol can serve as an effector for ubiquinone. The maximal binding capacity of α-[¹⁴C]tocopherol, dispersed in 50 mM phosphate buffer containing 0.25% sodium cholate, pH 7.4, to succinate-cytochrome c reductase was shown to be 0.68 μmol/mg protein. A similar binding capacity, based on cytochrome b content, was observed in submitochondrial particles. Binding of α-tocopherol to succinate-cytochrome c reductase not only caused an inhibition of enzymatic activity but also caused a reduction of cytochrome c₁ in the absence of substrate, a phenomenon analogous to the removal of phospholipids from the enzyme preparation. Furthermore, binding of α-tocopherol to succinate-cytochrome c reductase decreased the rate of reduction of cytochrome b by succinate. Since electron transfer from succinate to ubiquinone was not affected by α-tocopherol treatment, the decrease in reduction rate of cytochrome b by succinate must be due to a change in environment around cytochrome b. These results as well as the fact that reactivation of α-tocopherol-inhibited enzyme requires only low concentrations of ubiquinone were used to explain the inhibitory effect as a result of a change in protein conformation and protein-phospholipid interaction rather than the direct displacement of ubiquinone by α-tocopherol. This deduction was further supported by the fact that no ubiquinone was released from succinate-cytochrome c reductase upon treatment with α-tocopherol.     

The action of bothrops neuwiedii phospholipase A2 on mitochondrial phospholipids and electron transfer

Summary Incubation of heart-muscle mitochondrial fragments (Keilin-Hartree preparation, henceforth KH particles) withBothrops neuwiedii phospholipase A₂ ((EC 3.1.1.4); isoenzyme P-1;Vidal, J. C. et al., Arch. Biochem. Biophys. 151, 168 (1972)) in the presence of Ca²⁺, affected both the phospholipid content and catalytic properties of particles. Phospholipid analysis revealed the hydrolysis of phosphatidylcholine, phosphatidalcholine, phosphatidylethanolamine and phosphatidalethanolamine (with concomitant increase of lysophosphatides); diphosphatidyl glycerol (cardiolipin) was not attacked. Investigation of digested particles with adequate electron acceptors (or donors) showed inhibitions at the NADH-ubiquinone segment and the cytochromeb-c segment of the respiratory chain; a third less sensitive site was at (or near) cytochrome oxidase. In contrast to these inhibitory effects, the activities of succinate dehydrogenase and NADH-ferricyanide reductase were increased. The inhibition of electron transfer was due to the action of the products of phospholipid hydrolysis (unsaturated fatty acids and lysophosphatides) since (a), lipid extracts from phospholipase digested particles inhibited intact particles in much the same manner as phospholipase digestion; (b), unsaturated fatty acids inhibited NADH-oxidase and activated succinate and NADH-dehydrogenases; (c), lysophosphatidyl choline inhibited NADH- and succinate oxidases; (d), washing of digested particles with BSA completely restored NADH-oxidase activity; (e), after extraction of the digested lipids with aqueous acetone, rebinding of ubiquinone and non-digested lipid completely restablished electron transfer from succinate to cytochromec; (f) the content of ubiquinone, cytochromesb andc +c ₁ was not affected by phospholipase digestion. The summarized data taken together are similar to the effects ofB. neuwiedii andCrotalus adamanteus phospholipases on electron transfer particles. On the other hand, the contrast with theNaja naja phospholipase (s) was remarkable.Abbreviations KH particles Keilin-Hartree particles (or preparation) - LPC lysophosphatidyl choline - BSA bovine serum albumin - tlc thin layer chromatography - UQ_o 2,3-dimethoxy-5-methyl-1,4-benzoquinone - K₃H₂ menadiol - PMS phenazine methosulfate     

Antimycin-resistant alternate electron pathway to plastocyanin in bovine-heart Complex III

Bovine-heart Complex III can catalyze the reduction of spinach plastocyanin by a decyl analog of ubiquinol-2 at a rate comparable with the rate of plastocyanin reduction by plastoquinol as catalyzed by the cytochromeb ₆–f complex purified from spinach leaves. This plastocyanin reduction as catalyzed by Complex III was almost completely inhibited by myxothiazol at stoichiometric concentrations, partially inhibited by UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) and funiculosin, and was relatively insensitive to antimycin and HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide). Cytochromec reduction as catalyzed by Complex III displayed a residual, inhibitor-insensitive rate of 5% of the uninhibited rate for each of the three inhibitors, antimycin, myxothiazol, and UHDBT. However, the residual rate that was insensitive to each of the inhibitors added singly was inhibited further by addition of the remaining two inhibitors. From these results it is concluded that plastocyanin reduction involves an electron-transfer pathway through Complex III that is distinct from the pathway utilized for reduction of cytochromec.A portion of the data in this report was presented at the IV International Symposium on Coenzyme Q₁₀, Munich, F.R.G., November, 6–9, 1983.     

The site of inhibition of mitochondrial electron transfer by coenzyme Q analogues.

The effects of 33 quinone derivatives on mitochondrial electron transfer in yeast were examined. Twenty-two of the compounds were also tested for their effects on the growth of yeast cells. Four strong inhibitors of electron transfer were identified: 5-n-undecyl-6-hydroxy-4, 7-dioxobenzothiazole, 7-ω-cyclohexyloctyl-6-hydroxy-5,8-quinolinequinone, 7-n-hexadecyl-mercapto-6-hydroxy-5, 8-quinolinequinone, and 3-n-dodecylmercapto-2-hydroxy-1, 4-naphthoquinone. They inhibit the growth of yeast with ethanol as an energy source, but not when glucose is the energy source. The NADH oxidase activity of isolated mitochondria is 50% inhibited by these quinone derivatives at about 10^?8m, or 0.5 μmol/g mitochondrial protein; 1000-fold higher concentrations do not affect electron transfer from NADH or succinate to coenzyme Q₂. The effects of the inhibitors on cytochrome spectra indicate that they block electron transfer between cytochromes b and c₁. A possible antagonism between these compounds and coenzyme Q at a site between cytochromes b and C₁ is discussed in terms of Mitchell's “protonmotive Q cycle” hypothesis (Mitchell, P. (1976) J. Theor. Biol. 62, 327–367). 6-β-naphthylmercapto-5-chloro-2,3-dimethoxy-1,4-benzoquinone inhibits electron transfer between succinate and coenzyme Q₂ or phenazine methosulfate, suggesting a site in the succinate-coenzyme Q reductase complex with a different quinone specificity from that of the site in the cytochrome bc₁ complex. Seven of the quinone derivatives inhibit growth on both glucose and ethanol media, indicating that their effect is not the result of inhibition of respiration.     

Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter

Summary The substrate stoichiometry of the intestinal Na⁺/phosphate cotransporter was examined using two measures of Na⁺-dependent phosphate uptake: initial rates of uptake with [³²P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC₃(5). Isotopic phosphate measures electrogenic and electroneutral Na⁺-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na⁺/phosphate cotransport was found to have similar Na⁺ activations (apparentK _0.5's of 28 and 25mm), apparentK _m 's for phosphate (100 and 410 m), andK _0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na⁺-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na⁺ with apparentK _0.5 for phosphate of 200 m; similar to the apparentK _m for phosphate uptake, andK _0.5 for phosphate protection against FITC-PG inhibition of Na⁺-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na⁺/phosphate cotransporter is electrogenic at pH 7.4, that H₂PO ₄ ^– is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na⁺/phosphate cotransporter.     

Spectral and functional characterization of membrane fragments from the facultative photosynthetic bacterium Rhodopseudomonas blastica

The cytochromes of photosynthetically grown Rhodopseudomonas blastica have been thermodynamically characterized using the technique of redox titrations. Six cytochromes were present; two cytochromes c, E _m7= +295mV, E _m7=+345mV; and four cytochromes b, E _m7=+290mV, E _m7=+130mV, E _m7=+60mV, E _m7=-4mV. These cytochromes were tightly bound except for cytochrome c with E _m7 of+345mV which was mostly present in the soluble cell extracts.The effects of cyanide on both the cytochrome c oxidase activity and the NADH-dependent respiration, revealed the presence of a branched respiratory chain, one branch leading to a cyanide-resistant oxidase containing pathway and the other including the cyanide-sensitive cytochrome c-oxidase.The effects of antimycin A, myxothiazol and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) on the steadystate NADH-dependent respiration were also studied. Antimycin A and myxothiazol appeared to act at the level of the ubiquinol-cytochrome c oxidoreductase while UHDBT drastically affected both respiratory branches.Absorption spectra of chromatophore photopigments resulted to be similar to those reported in many species of facultative photosynthetic bacteria although carotenoid absorption maxima were blue-shifted by 5 nm.The light-induced oxygen reduction performed by chromatophores from R. blastica suggested a strict interaction between photosynthetic and respiratory apparatuses.     

Iron assimilation in plants: reduction of a ferriphytosiderophore by NADH:nitrate reductase from squash

NADH:nitrate reductase (EC 1.6.6.1) from squash (Cucurbita maxima Duch., cv. Buttercup) can catalyze the reduction of a ferriphytosiderophore from barley (Hordeum vulgare L. cv. Europa). Maximal activity occurs at pH 6, with an apparentK _m andV _max of 76 M and 21 nmol·min^-1·(mg protein)^-1, respectively. The ferriphytosiderophore strongly inhibits nitrate reduction catalyzed by nitrate reductase at the optimal pH for nitrate reduction, i.e. 7.5. On the contrary, nitrate is a poor inhibitor of ferriphytosiderophore reduction catalyzed by nitrate reductase at the optimal pH for this reaction, pH 6.0. Thus, squash has the potential to assimilate the iron from a ferriphytosiderophore synthesized by another plant.     

Effect of ubiquinone extraction on the reaction of the mitochondrialbc 1 complex with ferricyanide

Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochromeb extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochromec was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/molbc ₁ complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochromeb extrareduction. Since the initial rates of cytochromeb reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochromeb was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.     

Genetic Evidence for Coenzyme Q Requirement in Plasma Membrane Electron Transport

Plasma membranes isolated from wild-type Saccharomyces cerevisiae crude membrane fractions catalyzed NADH oxidation using a variety of electron acceptors, such as ferricyanide, cytochrome c, and ascorbate free radical. Plasma membranes from the deletion mutant strain coq3, defective in coenzyme Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q₆ and contained greatly diminished levels of NADH–ascorbate free radical reductase activity (about 10% of wild-type yeasts). In contrast, the lack of coenzyme Q₆ in these membranes resulted in only a partial inhibition of either the ferricyanide or cytochrome-c reductase. Coenzyme Q dependence of ferricyanide and cytochrome-c reductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones. Ascorbate free radical reductase was unique because it was highly dependent on coenzyme Q and did not involve superoxide since it was not affected by superoxide dismutase (SOD). Both coenzyme Q₆ and NADH–ascorbate free radical reductase were rescued in plasma membranes derived from a strain obtained by transformation of the coq3 strain with a single-copy plasmid bearing the wild type COQ3 gene and in plasma membranes isolated form the coq3 strain grown in the presence of coenzyme Q₆. The enzyme activity was inhibited by the quinone antagonists chloroquine and dicumarol, and after membrane solubilization with the nondenaturing detergent Zwittergent 3–14. The various inhibitors used did not affect residual ascorbate free radical reductase of the coq3 strain. Ascorbate free radical reductase was not altered significantly in mutants atp2 and cor1 which are also respiration-deficient but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase in coq3 mutants is related solely to the inability to synthesize ubiquinone and not to the respiratory-defective phenotype. For the first time, our results provide genetic evidence for the participation of ubiquinone in NADH–ascorbate free radical reductase, as a source of electrons for transmembrane ascorbate stabilization.     

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochromec oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochromec reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochromec oxidoreductase is strongly stimulated by added CoQ₃, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of theK _m for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ₉ and CoQ₁₀.     

Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter

Summary l-proline uptake via the intestinal brush-borderIMINO carrier was tested for inhibition by 41 compounds which included sugars, N-methylated, -,-, - and -amino and imino acids, and heterocyclic analogs of pyrrolidine, piperidine and pyridine. Based on competitive inhibitor constants (apparentK/'s) we find that theIMINO carrier binding site interacts with molecules which possess a well-defined set of structural prerequisites. The ideal inhibitor must 1) be a heterocyclic nitrogen ring, 2) have a hydrophobic region, 3) be thel-stereoisomer of 4) an electronegative carbonyl group which is 5) separated by a one-carbon atom spacer from 6) an electropositive tetrahedral imino nitrogen with two H atoms. Finally, 7) the inhibitor conformation determined by dynamic ring puckering must position all these features within a critical domain. The two best inhibitors arel-pipecolate (apparentK/0.2mm) andl-proline (apparentK/0.3mm).     

Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

Separation of respiratory reactions in Rhodospirillum rubrum: Inhibition studies with 2-hydroxydiphenyl

1. Respiration of chemotrophically and phototrophically grown Rhodospirillum rubrum is inhibited by 2-hydroxydiphenyl.2. Membrane-bound NADH oxidase and NADH: cytochrome c reductase are inhibited also. The inhibitor constant for both reactions (K_i) is 0.075±0.012 mM. NADH dehydrogenase is not inhibited significantly.3. The inhibition of succinate:cytochrome c reductase is associated for chemotrophic membranes with K_i = 0.22±0.03 mM and for phototrophic membranes with K_i = 0.49±0.09 mM. Succinate dehydrogenase is not affected by 2-hydroxydiphenyl.4. Cytochrome oxidase is inhibited only slightly.5. While NADH-dependent reactions in both phototrophic and chemotrophic membranes are inhibited maximally more than 95%, succinate-dependent reactions can be inhibited more than 95% only in chemotrophic membranes. In photo-trophic membranes the maximum inhibition of succinate-dependent reactions is about 70%.6. The type of inhibition in both cases 2 and 3 is non-competitive.7. While the reduction of b-type cytochrome is inhibited by 2-hydroxydiphenyl, the degree of ubiquinone reduction is not influenced. The data suggest that the site of inhibition is localized between ubiquinone and cytochrome b.8. Implications of these data for the respiratory electron transport system in R. rubrum are discussed.