The aerobic electron transport system of Eikenella corrodens

The respiratory system of the fastidious beta-proteobacterium Eikenella corrodens grown with limited oxygen was studied. Membranes showed the highest oxidase activity with ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) or succinate and the lowest activity with NADH and formate. The presence of a bc1-type complex was suggested by the inhibition exerted by 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), myxothiazol, and antimycin A on respiration with succinate and by the effect of the latter two inhibitors on the succinate-reduced difference spectra. Respiration with succinate or ascorbate-TMPD was abolished by low KCN concentrations, suggesting the presence of a KCN-sensitive terminal oxidase. Cytochromes b and c were spectroscopically detected after reduction with physiological or artificial electron donors, whereas type a and d cytochromes were not detected. The CO difference spectrum of membranes reduced by dithionite and its photodissociation spectrum (77 K) suggested the presence of a single CO compound that had the spectral features of a cytochrome o-like pigment. High-pressure liquid chromatography analysis of membrane haems confirmed the presence of haem B; in contrast, haems A and O were not detected. Peroxidase staining of membrane type c cytochromes using SDS-PAGE revealed the presence of five bands with apparent molecular masses of 44, 33, 30, 26, and 14 kDa. Based on our results, a tentative scheme of the respiratory chain in E. corrodens, comprising (i) dehydrogenases for succinate, NADH, and formate, (ii) a ubiquinone, (iii) a cytochrome bc1, and (iv) a type-cbb' cytochrome c oxidase, is proposed.     

Funiculosin: an antibiotic with antimycin-like inhibitory properties.

The antibiotic funiculosin mimics the action of antimycin in several ways. It inhibits the oxidation of NADH and succinate, but not TMPD+ascorbate. The titer for maximal inhibition in Mg2+-ATP particles (0.4-0.6 nmol/mg protein) is close to the concentrations of cytochromes b and cc1. Funiculosin also induces the oxidation of cytochromes cc1 and an extra reduction of cytochrome b in the aerobic steady state, and it inhibits duroquinol-cytochrome c reductase activity in isolated Complex III. The location of the funiculosin binding site is clearly similar to that of antimycin. In addition, funiculosin, like antimycin, prevents electron transport from duroquinol to cytochrome b in isolated Complex III if the complex is pre-reduced with ascorbate. Funiculosin and antimycin differ, however, in the manner in which they modulate the reduction of cytochrome b by ascorbate+TMPD.     

Reconstitution of biological molecular generators of electric current. Cytochrome oxidase.

1. Direct measurement of the electric current generation by cytochrome oxidase has been carried out. To this end, two procedures were used. The simpler one consists in formation of planar artificial membrane from the mixture of decane solution of soya bean phospholipids and beef heart cytochrome oxidase. Addition of cytochrome c and ascorbate to one of the two compartments separated by the cytochrome oxidase-containing planar membrane was found to result in a transmembrane electric potential difference being formed (plus on cytochrome c side of the membrane). Maximal values of potential differences obtained by this method were about 40 mV. Much higher potentials were observed when another ("photeoliposome-planar membrane") method was applied. In this case cytochrome oxidase was reconstituted with phospholipid to form proteoliposomes which adhered to planar phospholipid membrane in the presence of Ca2+ ions. Addition of cytochrome c and ascorbate to the proteoliposome-containing compartment gives rise to generation of an electric potential difference across the planar membrane, which reached 100 mV at a current of about 1 X 10(-11) A (minus in the proteoliposome-free compartment). The electromotive force of this generator was estimated as being about 0.2 V. If ascorbate and proteoliposomes were added into different compartments, a penetrating hydrogen atom carrier (phenazine methosulfate, (PMS) or tetramethyl-p-phenylenediamine (TMPD)) was required for a membrane potential to be formed. Generation of an electric potential difference of the opposite direction (plus in the proteoliposome-free compartment) was revealed in experiments with cytochrome oxidase proteoliposome containing cytochrome c in their interior. In this case, addition of PMS or TMPD was necessary. 2. In the suspension of cytochrome oxidase proteoliposome the uptake of a cationic penetrant (tetraphenyl phosphonium cation) was found to be coupled with electron transfer via external cytochrome c. Electron transfer via intraproteoliposomal cytochrome c induced the uptake of anionic penetrants (tetraphenyl borate and phenyldicarbaundecaborane anions). 3. All the above effects were sensitive to cyanide and protonophorous uncouplers. 4. In proteoliposomes containing both cytochrome oxidase and bacteriorhodopsin, the light- and oxidation-dependent generations of membrane potential have been revealed. 5. The data obtained are in agreement with Mitchell's idea of transmembrane electron flow in the cytochrome oxidase segment of the respiratory chain.     

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl- p -phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa ₃. In PMS-treated eggs and fertilized eggs, the maximum photoreactivation of CO-inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO-inhibited respiration in TMPD-treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b , which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.     

Role of menaquinone in inactivation and activation of the Bacillus cereus forespore respiratory system.

The respiratory systems of the Bacillus cereus mother cell, forespore, and dormant and germinated spore were studied. The results indicated that the electron transfer capacity during sporulation, dormancy, and germination is related to the menaquinone levels in the membrane. During the maturation stages of sporulation (stages III to VI), forespore NADH oxidase activity underwent inactivation concomitant with a sevenfold decrease in the content of menaquinone and without major changes in the content of cytochromes and segment transfer activities. During the same period, NADH oxidase and menaquinone levels in the mother cell compartment steadily decreased to about 50% at the end of stage VI. Dormant spore membranes contained high levels of NADH dehydrogenase and cytochromes, but in the presence of NADH, they exhibited very low levels of O2 uptake and cytochrome reduction. Addition of menadione to dormant spore membranes restored NADH-dependent respiration and cytochrome reduction. During early germination, NADH-dependent respiration and cytochrome reduction were restored simultaneously with a fourfold increase in the menaquinone content; during germination, no significant changes in cytochrome levels or segment electron transfer activities of the respiratory system took place.     

Kinetics of cytochrome c and TMPD oxidation by cytochrome c oxidase from the thermophilic bacterium, PS3

Cytochrome caa3 (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes. It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes. Proteoliposomal cytochrome caa3 develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c. Reduction of the a3 centre is blocked in the presence of cyanide. During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms. During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated CuA atom are reduced next. It is concluded that, despite its size and hydrophobic association with the aa3 complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.     

Funiculosin: An antibiotic with antimycin-like inhibitory properties

The antibiotic funiculosin mimics the action of antimycin in several ways. It inhibits the oxidation of NADH and succinate, but not TMPD+ascorbate. The titer for maximal inhibition in Mg²⁺-ATP particles (0.4–0.6 nmol/mg protein) is close to the concentrations of cytochromes b and cc₁. Funiculosin also induces the oxidation of cytochromes cc₁ and an extra reduction of cytochrome b in the aerobic steady state, and it inhibits duroquinol-cytochrome c reductase activity in isolated Complex III. The location of the funiculosin binding site is clearly similar to that of antimycin. In addition, funiculosin, like antimycin, prevents electron transport from duroquinol to cytochrome b in isolated Complex III if the complex is pre-reduced with ascorbate. Funiculosin and antimycin differ, however, in the manner in which they modulate the reduction of cytochrome b by ascorbate+TMPD.     

Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: effects of procedural manipulations

Differences in oxidative metabolism between subsarcolemmal and interfibrillar heart mitochondria were investigated. Interfibrillar mitochondria oxidized substrates donating reducing equivalents at Complex I (NADH-CoQ reductase), Complex II (succinate-CoQ reductase), and Complex III (CoQH2-cytochrome c reductase) more rapidly than did subsarcolemmal mitochondria. There was no difference in oxidation of substrates entering the electron transport chain at Complex IV (cytochrome c oxidase). Differences expressed in normal-ionic-strength medium at Complexes II and III but not I were eliminated in low-ionic-strength medium. The concentrations of cytochromes and activities of NADH and cytochrome c oxidase were virtually the same in the two populations. In permeabilized mitochondria, activities of succinate-duroquinone and TMPD plus ascorbate oxidase were significantly lower in the subsarcolemmal mitochondria. Differences in membrane permeability between the populations were suggested by the greater permeability of subsarcolemmal mitochondria to exogenous NADH. The influence of isolation buffers and preparative procedures on the two classes of mitochondria were also examined. Characteristic biochemical and morphological properties of the two populations were unchanged by exposing each to the preparative procedure used to isolate the alternate population; the oxidative performance of the two populations cannot be equalized by experimental manipulation.     

Inhibition of electron transfer from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone by antimycin.

The effect of antimycin on (i) the respiratory activity of the KCN-insensitive pathway of mitochondria of Neurospora grown on chloramphenicol (chloramphenicol-grown) with durohydroquinone and succinate or NADH as substrate, (ii) the electron transfer from the b-type cytochromes to ubiquinone with durohydroquinone as electron donor as well as (iii) the electron transfer from the b-type cytochromes to duroquinone with succinate as electron donor in chloramphenicol-grown Neurospora and beef heart submitochondrial particles was studied. All experiments were performed in the uncoupled state. 1. The respiratory chain of chloramphenicol-grown Neurospora mitochondria branches at ubiquinone into two pathways. Besides the cytochrome oxidase-dependent pathway, a KCN-insensitive branch equiped with a salicylhydroxamate-sensitive oxidase exists. Durohydroquinone, succinate or NADH are oxidized via both pathways. The durohydroquinone oxidation via the KCN-insensitive pathway is inhibited by antimycin, wheras the succinate or NADH oxidation is not. The titer for ful inhibition is one mol antimycin per mol cytochrome b-563 or cytochrome b-557. 2. The electron transfer from durohydroquinone to ubiquinone, which takes place in the KCN-inhibited state, does not occur in the antimycin-inhibited state. 3. The reduction of duroquinone by succinate in the presence of KCN is inhibited by antimycin. The titer for full inhibition is one mol antimycin per mol cytochrome b-566 or cytochrome b-562 for beef heart (or cytochrome b-563 or cytochrome b-557 for Neurospora). 4. When electron transfer from the b-type cytochromes to cytochrome C1, ubiquinone and duroquinone is inhibited by antimycin, the hemes of cytochrome b-566 and cytochrome b-562 (or cytochrome b-563 and cytochrome b-557) are in the reduced state. 5. The experimental results suggest that the two b-type cytochromes form a binary complex the electron transferring activity of which is inhibited by antimycin, the titer for full inhibition being one mol of antimycin per mol of complex. The electron transfer from the b-type cytochromes to ubiquinone is inhibited in a non-linear fashion.     

Tn5-induced cytochrome mutants of Bradyrhizobium japonicum: effects of the mutations on cells grown symbiotically and in culture.

Two Bradyrhizobium japonicum cytochrome mutants were obtained by Tn5 mutagenesis of strain LO and were characterized in free-living cultures and in symbiosis in soybean root nodules. One mutant strain, LO501, expressed no cytochrome aa3 in culture; it had wild-type levels of succinate oxidase activity but could not oxidize NADH or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The cytochrome content of LO501 root nodule bacteroids was nearly identical to that of the wild type, but the mutant expressed over fourfold more bacteroid cytochrome c oxidase activity than was found in strain LO. The Tn5 insertion of the second mutant, LO505, had a pleiotropic effect; this strain was missing cytochromes c and aa3 in culture and had a diminished amount of cytochrome b as well. The oxidations of TMPD, NADH, and succinate by cultured LO505 cells were very similar to those by the cytochrome aa3 mutant LO501, supporting the conclusion that cytochromes c and aa3 are part of the same branch of the electron transport system. Nodules formed from the symbiosis of strain LO505 with soybean contained no detectable amount of leghemoglobin and had no N2 fixation activity. LO505 bacteroids were cytochrome deficient but contained nearly wild-type levels of bacteroid cytochrome c oxidase activity. The absence of leghemoglobin and the diminished bacterial cytochrome content in nodules from strain LO505 suggest that this mutant may be deficient in some aspect of heme biosynthesis.     

The cytochrome cbo from the obligate methylotroph Methylobacillus flagellatus KT is a cytochrome-c oxidase

The oxidase cho of Methylobacillus flagellatus KT was purified to homogeneity by nondenaturing gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cbo with a pH optimum of 8.3. When TMPD served as electron donor for the oxidase cho, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and of only ascorbate. The kinetic constants, determined at pH 7.0, were as follows: oxidation by the enzyme of reduced TMPD at pH 7.0 was characterized by KM = 0.86 mM and Vmax = 1.1 mumol O2/(min mg protein), and oxidation of reduced cytochrome c from horse heart was characterized by KM = 0.09 mM and Vmax = 0.9 mumol O2/(min mg protein) Cyanide inhibited ascorbate/TMPD oxidase activity (Ki = 4.5-5.0 microM). The soluble cytochrome cH (12 kDa) partially purified from M. flagellatus KT was found to serve as the natural electron donor for the oxidase cbo.     

Respiratory Mechanisms in the Flexibacteriaceae: Terminal Oxidase Systems of Saprospira grandis and Vitreoscilla Species

Particles from both Saprospira grandis and Vitreoscilla species, obtained by high-pressure extrusion and sonic treatment, respectively, actively catalyze the oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate with O(2). These activities are inhibited by cyanide but not by antimycin; Saprospira is also amytal- and rotenone-insensitive. Vitreoscilla preparations were unable to oxidize mammalian ferrocytochrome c and reduced tetramethyl-p-phenylenediamine, whereas the Saprospira preparations did so actively. Low-temperature (77 K) difference spectroscopy of Vitreoscilla cells and particles indicates the presence of three maxima in the cytochrome alpha-region at 554, 558, and 562 nm. All three cytochromes are active in NADH and succinate oxidation, but none is ascorbate reducible. Cytochrome o is the only CO-binding pigment present and is probably the terminal oxidase; it has properties similar to the cytochrome o isolated in solubilized form from this organism. Saprospira cells and membranes exhibit four cytochrome absorption bands whose maxima are at 550, 554, 558, and 603 nm at 77 K. The latter component has not been noted previously. NADH and succinate reduce all four cytochromes, but ascorbate reduces only the 550- and 603-nm pigments. CO spectra indicate the presence of cytochrome a,a(3) which is probably the oxidase. A second CO-binding pigment is present which is not a peroxidase but may be a cytochrome.     

Periplasmic oxygen reduction by Desulfovibrio species

Washed cells of Desulfovibrio vulgaris strain Marburg (DSM 2119) reduced oxygen to water with H(2) as electron donor at a mean rate of 253 nmol O(2) min(-1) (mg protein)(-1). After separating the periplasm from the cells, more than 60% of the cytochrome c activity and 90% of the oxygen-reducing activity were found in the periplasmic fraction. Oxygen reduction and the reduction of cytochrome c with H(2) were inhibited by CuCl(2). After further separation of the periplasm by ultrafiltration (exclusion sizes 30, 50, and 100 kDa), oxygen reduction with H(2) occurred with the retentates only. Ascorbate plus tetramethyl-p-phenylenediamine (TMPD), however, were also oxidized by the filtrates. The stoichiometry of 1 mol O(2) reduced per 2 mol ascorbate oxidized indicated the formation of water. Our experiments present evidence that in D. vulgaris periplasmic hydrogenase and cytochrome c play a major role in oxygen reduction. Preliminary studies with other Desulfovibrio species indicated a similar function of periplasmic c-type cytochromes in D. desulfuricans CSN and D. termitidis KH1.     

The respiratory electron transport system of heterotrophically-grown Rhodopseudomonas palustris.

The enzymatic activities and the cytochrome components of the respiratory chain were investigated with membrane fractions from chemoheterotrophically growth Rhodopseudomonas palustris. Whereas the level of electron transfer carriers was not distinctly affected by a change of the culture conditions, the potential activities of the enzymes were clearly increased when the cells were grown aerobically. Reduced-minus oxidized difference spectra of the membrane fractions prepared from dark aerobically grown cells revealed the presence of three beta-types cytochromes b561, b560 and b558, and at least two c-type cytochromes c556 and c2 as electron carriers in the electron transfer chain. Cytochrome of a-type could not be detected in these membranes. Reduced plus CO minus reduced difference spectra of the membrane fractions were indicative of cytochrome o, which may be equivalent to cytochrome b560, appearing in substrate-reduced minus oxidized difference spectra. Cytochrome o was found to be the functional terminal oxidase. CO difference spectra of the high speed supernatant fraction indicated the presence of cytochrome c'. Succinate and NADH reduced the same types of cytochromes. However, a considerable amount of cytochrome b561 with associated beta and gamma bands at 531 and 429 nm, respectively, was reducible by succinate, but not by NADH. A substantial fraction of the membrane-bound b-type cytochrome was non-substrate reducible and was found in dithionite-reduced minus substrate-reduced spectra. Cytochrome c2 may be localized in a branch of the electron transport system, with the branch-point at the level of ubiquinone. The separate pathways rejoined at a common terminal oxidase. Two terminal oxidases with different KCN sensitivity were present in the respiratory chain, one of which was sensitive to low concentrations of KCN and was connected with the cytochrome chain. The other terminal oxidase which was inhibited only by high concentrations of cyanide was located in a branched pathway, through which the electrons could flow from ubiquinone to oxygen bypassing the cytochrome chain.     

Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.     

The oxidative activities of membrane vesicles from Bacillus caldolyticus. Energy-dependence of succinate oxidation

1. The properties of membrane vesicles from the extreme thermophile Bacillus caldolyticus were investigated. 2. Vesicles prepared by exposure of spheroplasts to ultrasound contained cytochromes a, b and c, and at 50 degrees C they rapidly oxidized NADH and ascorbate in the presence of tetramethyl-p-phenylenediamine. Succinate and l-malate were oxidized more slowly, and dl-lactate, l-alanine and glycerol 1-phosphate were not oxidized. 3. In the absence of proton-conducting uncouplers the oxidation of NADH was accompanied by a net translocation of H(+) into the vesicles. Hydrolysis of ATP by a dicyclohexylcarbodi-imide-sensitive adenosine triphosphatase was accompanied by a similarly directed net translocation of H(+). 4. Uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone or valinomycin plus NH(4) (+)) prevented net H(+) translocation but stimulated ATP hydrolysis, NADH oxidation and ascorbate oxidation. The last result suggested an energy-conserving site in the respiratory chain between cytochrome c and oxygen. 5. Under anaerobic conditions the reduction of cytochrome b by ascorbate (with tetramethyl-p-phenylenediamine) was stimulated by ATP hydrolysis, indicating an energy-conserving site between cytochrome b and cytochrome c. However, no reduction of NAD(+) supported by oxidation of succinate, malate or ascorbate occurred, neither did it with these substrates in the presence of ATP under anaerobic conditions, suggesting that there was no energy-conserving site between NADH and cytochrome b. 6. Succinate oxidation, in contrast with that of NADH and ascorbate, was strongly inhibited by uncouplers and stimulated by ATP hydrolysis. These effects were not observed when phenazine methosulphate, which transfers electrons from succinate dehydrogenase directly to oxygen, was present. It was concluded that in these vesicles the oxidation of succinate was energy-dependent and that the reoxidation of reduced succinate dehydrogenase was dependent on the outward movement of H(+) by the protonmotive force. 7. In support of the foregoing conclusion it was shown that the reduction of fumarate by NADH was an energy-conserving process. 8. If the activities of vesicles accurately represent those of the intact organism it appears that in B. caldolyticus the reduction of fumarate to succinate at the expense of reducing equivalents from NADH is energetically favoured over succinate oxidation even under aerobic conditions. This may be related to the need for an ample supply of succinate for haem synthesis in order to provide cytochromes for the organism.     

Ubiquinol:cytochrome c oxidoreductase (complex III). Effect of inhibitors on cytochrome b reduction in submitochondrial particles and the role of ubiquinone in complex III

Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c(1)) of complex III, addition of succinate reduced heme b(H) followed by a slow and partial reduction of heme b(L). Similar results were obtained when SMP were treated only with KCN or NaN(3), reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of b(H) reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both b(H) and b(L) were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c(1) on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing 相似文献   

Simultaneous presence of two terminal oxidases in the respiratory system of dark aerobically grown Rhodospirillum rubrum

Rhodospirillum rubrum CAF10, a spontaneous cytochrome oxidase defective mutant, was isolated from strain S1 and used to analyze the aerobic respiratory system of this bacterium. In spite of its lack of cytochrome oxidase activity, strain CAF10 grew aerobically in the dark although at a decreased rate and with a reduced final yield. Furthermore, aerobically grown mutant cells took up O₂ at high rates and membranes isolated from those cells exhibited levels of NADH and succinate oxidase activities which were similar to those of wild type membranes. It was observed also that whereas in both strains O₂ uptake (intact cells) and NADH and succinate oxidase activities (isolated membranes) were not affected by 0.2 mM KCN, the cytochrome oxidase activity of the wild type strain was inhibited about 90% by 0.2 mM KCN. These data indicate the simultaneous presence of two terminal oxidases in the respiratory system of R. rubrum, a cytochrome oxidase and an alternate oxidase, and suggest that the rate of respiratory electron transfer is not limited at the level of the terminal oxidases. It was also found that the aerobic oxidation of cellular cytochrome c ₂ required the presence of a functional cytochrome oxidase activity. Therefore it seems that this electron carrier, which only had been shown to participate in photosynthetic electron transfer, is also a constituent of the respiratory cytochrome oxidase pathway.Abbreviations DCIP 2,6-dichlorophenolindophenol - DMPD N,N-dimethyl-p-phenylenediamine - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]-glycine     

Sodium-ion stimulated amino acid uptake in membrane vesicles of alkalophilic Bacillus no. 8-1

Membrane vesicles were isolated from alkalophilic Bacillus No. 8-1, and the active transport of amino acids was studied. The transport of amino acids was dependent upon substrate oxidation and the presence of Na+. Concentrative uptake of amino acids was stimulated by the addition of an artificial electron donor system, ascorbate-phenazine methosulfate (PMS), and to a lesser extent by NADH, while succinate, L-lactate, and alpha-glycerol-phosphate did not stimulate the uptake. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) and cytochrome c were able to replace PMS, and reduced forms of these compounds were also very efficient electron donors. Amino acid transport was dependent on electron transfer, and inhibition of NADH oxidation by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), and sodium azide directly prohibited serine transport. The pH optima for serine transport lay between pH 8 and 9 for all energy sources. Sodium ion stimulated serine transport in the presence of NADH, NADH plus cytochrome c or succinate plus PMS, but had no stimulatory effect on the corresponding dehydrogenase activities. Sodium ion was also required for accumulation of serine in response to an artificial membrane potential where the respiratory chain was not operative. These results indicated that the stimulatory effect of Na+ on amino acid uptake was on the transport process itself.     

Construction and characterization of a mutant of alkaliphilic Bacillus firmus OF4 with a disrupted cta operon and purification of a novel cytochrome bd.

The caa3-type terminal oxidase of Bacillus firmus OF4 has been proposed to play an important role in the growth and bioenergetics of this alkaliphile (A. A. Guffanti and T. A. Krulwich, J. Biol. Chem. 267:9580-9588, 1992). A mutant strain was generated in which the cta operon encoding the oxidase was disrupted by insertion of a spectinomycin resistance cassette. The mutant was unable to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). Absorption spectra of membranes confirmed the loss of the enzyme and indicated the presence of a cytochrome bd-type terminal oxidase. The mutant could grow on glucose but was unable to grow on malate or other nonfermentative carbon sources, despite the presence of the cytochrome bd. The cytochrome bd was purified from the mutant. The enzyme consisted of two subunits and, with menadiol as substrate, consumed oxygen with a specific activity of 12 micromol of O2 x min(-1) x mg(-1). In contrast to both cytochromes bd of Escherichia coli, the enzyme did not utilize TMPD as an electron source. A number of additional features, including subunit size and spectral properties, distinguish this cytochrome bd from its counterparts in E. coli and Azotobacter vinelandii.