The biogenesis of c-type cytochromes in Escherichia coli requires a membrane-bound protein, DipZ, with a protein disulphide isomerase-like domain

A mutant of Escherichia coli K-12, JCB606, which lacks all five c-type cytochromes synthesized during anaerobic growth in the presence of nitrite or tri-methylamine-N-oxide (TMAO), was totally defective in Nrf activity and also partially defective in TMAO reductase activity. The mutation in strain JCB606 was shown to affect expression of the tor operon, which contributes almost equally with the products of the dms operon to the rate of TMAO reduction by bacteria during anaerobic growth in the presence of TMAO. The mutation in strain JCB606, dipZ, was mapped by P1 transduction close to the mel operon at co-ordinate 4425 on the E. coli chromosome, the gene order being nrf–fdhF–mel–dipZ–ampC. Recombinant plasmids that restored Nrf activity to test-tube cultures of the mutant were isolated from a cosmid library. A 2.7 kb EcoRV–Smal fragment (co-ordinates 4443 to 4446 kb on the physical map of the E. coli chromosome) was found potentially to encode three genes arranged in at least two operons. The second gene, dipZ, was sufficient to complement the JCB606 mutation. The translated DNA sequence predicts that DipZ is a 53kDa integral membrane protein with a 37kDa N-terminal domain including at least six membrane-spanning helices and a 16kDa carboxy-terminal hydrophilic domain which includes a protein disulphide isomerase-like motif. It is suggested that DipZ is essential for maintaining cytochrome c apoproteins in the correct conformations for the covalent attachment of haem groups to the appropriate pairs of cysteine residues.     

The dependence on quinone specificity of terminal electron transport of bacteria

Bacterial quinones were extracted with pentane, and homologues or other quinones were reincorporated. In spite of the redox potential difference of 110 mV, menaquinone and demethylmenaquinone could replace each other in aerobic electron transport and fumarate respiration ofHaemophilus influenzae RAMC 18 Bensted andProteus mirabilis Harding & Nicholson. The enzymes involved may recognize the naphthoquinone structure and are not specific for menaquinone or demethylmenaquinone. Ubiquinone was not replaced in aerobic electron transport by naphthoquinones withPseudomonas fluorescens 28/5 Rhodes orAcinetobacter sp. 661/60 Mannheim, probably owing to the specificity for benzoquinones of the enzymes involved, since the redox potential difference between demethylmenaquinone and ubiquinone is only 76 mV.Haemophilus parainfluenzae 429 Pittman, which resembles aerobic bacteria with respect to the terminal electron transport system, could incorporate demethylmenaquinone or menaquinone. This organism seems to be defective in the synthesis of naphthoquinones but possesses the enzyme system for fumarate respiration.Haemophilus influenzae RAMC 18 Bensted, which produces only demethylmenaquinone, seems to be defective in synthesizing ubiquinone, but it also possesses the enzymes for a ubiquinonemediated aerobic respiration.     

An Escherichia coli mutant containing only demethylmenaquinone,but no menaquinone: effects on fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate respiration

The mutant strain AN70 (ubiE) of Escherichia coli which is known to lack ubiquinone (Young IG et al. 1971), was analyzed for menaquinone (MK) and demethylmenaquinone (DMK) contents. In contrast to the wild-type, strain AN70 contained only DMK, but no MK. The mutant strain was able to grow with fumarate, trimethylamine N-oxide (TMAO) and dimethylsulfoxide (DMSO), but not with nitrate as electron acceptor. The membranes catalyzed anaerobic respiration with fumarate and TMAO at 69 and 74% of wild-type rates. DMSO respiration was reduced to 38% of wild-type activities and nitrate respiration was missing (8% of wild-type), although the respective enzymes were present in wild-type rates. The results complement earlier findings which demonstrated a role for DMK only in TMAO respiration (Wissenbach et al. 1990). It is concluded, that DMK (in addition to MK) can serve as a redox mediator in fumarate, TMAO and to some extent in DMSO respiration, but not in nitrate respiration. In strain AN70 (ubiE) the lack of ubiquinone (Q) is due to a defect in a specific methylation step of Q biosynthesis. Synthesis of MK from DMK appears to depend on the same gene (ubiE).Abbreviations DMSO = dimethylsulfoxide - DMS = dimethylsulfide - TMAO = trimethylamine N-oxide - TMA = trimethylamine - BV = benzylviologen - BV_red = reduced benzylyiologen - Q = ubiquinone - MK = menaquinone - DMK = demethylmenaquinone - NQ = naphthoquinone     

Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains in Escherichia coli K12

 The assembly of anaerobically induced electron transfer chains in Escherichia coli strains defective in periplasmic disulphide bond formation was investigated. Strains deficient in DsbA, DsbB or DipZ (DsbD) were unable to catalyse formate-dependent nitrite reduction (Nrf activity) or synthesize any of the known c-type cytochromes. The Nrf⁺ activity and cytochrome c content of mutants defective in DsbC, DsbE or DsbF were similar to those of the parental, wild-type strain. Neither DsbC expressed from a multicopy plasmid nor a second mutation in dipZ (dsbD) was able to compensate for a dsbA mutation by restoring nitrite reductase activity and cytochrome c synthesis. In contrast, only the dsbB and dipZ (dsbD) strains were defective in periplasmic nitrate reductase activity, suggesting that DsbB might fulfil an additional role in anaerobic electron transport. Mutants defective in dipZ (dsbD) were only slightly more sensitive to Cu⁺⁺ ions at concentrations above 5 mM than the parental strain, but strains defective in DsbA, DsbB, DsbC, DsbE or DsbF were unaffected. These results are consistent with our earlier proposals that DsbA, DsbB and DipZ (DsbD) are part of the same pathway for ensuring that haem groups are attached to the correct pairs of cysteine residues of apocytochromes c in the E. coli periplasm. However, neither DsbE nor DsbF are essential for the reduction of DipZ (DsbD). Received: 28 February 1996 / Accepted: 5 June 1996     

The specific functions of menaquinone and demethylmenaquinone in anaerobic respiration with fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate by Escherichia coli

The respiratory activities of E. coli with H₂ as donor and with nitrate, fumarate, dimethylsulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as acceptor were measured using the membrane fraction of quinone deficient strains. The specific activities of the membrane fraction lacking naphthoquinones with fumarate, DMSO or TMAO amounted to 2% of those measured with the membrane fraction of the wild-type strain. After incorporation of vitamin K₁ [instead of menaquinone (MK)] into the membrane fraction deficient of naphthoquinones, the activities with fumarate or DMSO were 92% or 17%, respectively, of the activities which could be theoretically achieved. Incorporation of demethylmenaquinone (DMK) did not lead to a stimulation of the activities of the mutant. In contrast, the electron transport activity with TMAO was stimulated by the incorporation of either vitamin K₁ or DMK. Nitrate respiration was fully active in membrane fractions lacking either naphthoquinones or Q, but was 3% of the wild-type activity, when all quinones were missing. Nitrate respiration was stimulated on the incorporation of either vitamin K₁ or Q into the membrane fraction lacking quinones, while the incorporation of DMK was without effect. These results suggest that MK is specifically involved in the electron transport chains catalyzing the reduction of fumarate or DMSO, while either MK or DMK serve as mediators in TMAO reduction. Nitrate respiration requires either Q or MK.Abbreviations DMK demethylmenaquinone - MK menaquinone - Q ubiquinone - DMSO dimethylsulfoxide - TMAO trimethylamine N-oxide - DMS dimethylsulfide - TMA trimethylamine - BV benzylviologen     

Kinase Activity of ArcB from Escherichia coli Is Subject to Regulation by Both Ubiquinone and Demethylmenaquinone

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in ‘respiratory’ electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.     

Cytochrome-c reductases from wild-type and mutant strains of Aspergillus nidulans

Summary The sedimentation coefficients of the NADPH: cytochrome-c oxidoreductase enzymes from wild-type and mutant strains of Aspergillus nidulans have been estimated by sucrose density gradient centrifugation. In the wild-type, two species of cytochrome-c reductase were found, with sedimentation coefficients of 13.7s and 7.6s respectively. The 13.7s species did not appear to be associated with the enzymes of nitrate reduction, whereas the 7.6s species was closely associated with NADPH: nitrate oxidoreductase. In mutant strains lacking nitrate reductase, a thir species of cytochrome-c reductase with a sedimentation coefficient of 4.5s was found. There is some evidence that this 4.5s cytochrome-c reductase is a subunit or breakdown product of nitrate reductase and a model is presented for the role of this 4.5s cytocnorome-c reductase in the assembly of the intact nitrate reductase molecule.     

Fumarate reduction systems in members of the family Rhodospirillaceae with different quinone types

Nineteen established and one undesignated species of the Rhodospirillaceae were examined for fumarate reduction in connection with their quinone systems. The fumarate reductase activity with reduced methyl viologen (MVH) or FMNH₂ as electron donor was found in membrane (chromatophore) preparations from phototrophically grown cells of all species containing menaquinone (MK) and/or rhodoquinone. The species having ubiquinone as the sole quinone contained no fumarate reductase activity, except some Rhodobacter species showing the FMNH₂-dependent activity. The MVH-fumarate reductase activity of the MK-type species was not inhibited by Triton X-100 or acetone treatment, suggesting the presence of a fumarate reductase reacting directly with MVH, while such an enzyme was absent in the MK-lacking strains, with few exceptions. The FMNH₂-fumarate reduction system was abolished by a detergent or acetone extraction in all bacteria but differed much among species with different quinone types as to the response to respiratory inhibitors. These differences in fumarate-reducing properties and quinone systems among the phototrophic bacteria are discussed from evolutionary and taxonomic viewpoints.Non-standard abbreviations RQ rhodoquinone - MK menaquinone - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - TTFA 2-thenoyltrifluoroacetone     

Quinones as hydrogen carriers for a late step in anaerobic heme biosynthesis in Escherichia coli

A late step in anaerobic heme synthesis, the oxidation of protoporphyrinogen with fumarate as electron acceptor, was studied in extracts and particles of Escherichia coli mutants deficient in quinones or cytochromes. Mutants specifically deficient in menaquinone did not couple protoporphyrinogen oxidation to fumarate reduction, whereas mutants containing menaquinone but deficient in either ubiquinone or cytochromes exhibited this activity. These findings indicate that this coupled reaction is dependent upon menaquinone as hydrogen carrier but independent of ubiquinone and cytochromes. Other characteristics of this coupled reaction were also studied. The activity was located exclusively in the membrane fraction of cell-free extracts. Coproporphyrinogen III could not replace protoporphyrinogen as substrate. Methylene blue, triphenyl tetrazolium and nitrate, but not nitrite, could replace fumarate as anaerobic hydrogen acceptor. These findings have implications for the mechanism and regulation of microbial heme and chlorophyll synthesis and for the physiology of cytochrome synthesis in anaerobic microorganisms.     

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.     

Menaquinone (Vitamin K2) Biosynthesis: Localization and Characterization of the menA Gene from Escherichia coli

A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of a menA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from the orf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into the orf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.     

Role of quinones in electron transport to oxygen and nitrate in Escherichia coli. Studies with a ubiA− menA− double quinone mutant

A ubiA^? menA^? double quinone mutant of Escherichia coli K12 was constructed together with other isogenic strains lacking either ubiquinone or menaquinone. These strains were used to study the role of quinones in electron transport to oxygen and nitrate. Each of the four oxidases examined (NADH, d-lactate, α-glycerophosphate and succinate) required a quinone for activity. Ubiquinone was active in each oxidase system while menaquinone gave full activity in α-glycerophosphate oxidase, partial activity in d-lactate oxidase but was inactive in NADH and succinate oxidation. The aerobic growth rates, growth yields and products of glucose metabolism of the quinone-deficient strains were also examined. The growth rate and growth yield of the ubi⁺ menA^? strain was the same as the wild-type strain, whereas the ubiA^? men⁺ strain grew more slowly on glucose, had a lower growth yield (30% of wild type) and accumulated relatively large quantities of acetate and lactate. The growth of the ubiA^? menA^? strain was even more severely affected than that of the ubiA^? men⁺ strain.Electron transport from formate, d-lactate, α-glycerophosphate and NADH to nitrate was also highly dependent on the presence of a quinone. Either ubiquinone or menaquinone was active in electron transport from formate and the activity of the quinones in electron transport from the other substrates was the same as for the oxidase systems. In contrast, quinones were not obligatory carriers in the anaerobic formate hydrogenlyase system. It is concluded that the quinones serve to link the various dehydrogenases with the terminal electron transport systems to oxygen and nitrate and that the dehydrogenases possess a degree of selectivity with respect to the quinone acceptors.     

Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains in Escherichia coli K12

The assembly of anaerobically induced electron transfer chains in Escherichia coli strains defective in periplasmic disulphide bond formation was investigated. Strains deficient in DsbA, DsbB or DipZ (DsbD) were unable to catalyse formate-dependent nitrite reduction (Nrf activity) or synthesize any of the known c-type cytochromes. The Nrf⁺ activity and cytochrome c content of mutants defective in DsbC, DsbE or DsbF were similar to those of the parental, wild-type strain. Neither DsbC expressed from a multicopy plasmid nor a second mutation in dipZ (dsbD) was able to compensate for a dsbA mutation by restoring nitrite reductase activity and cytochrome c synthesis. In contrast, only the dsbB and dipZ (dsbD) strains were defective in periplasmic nitrate reductase activity, suggesting that DsbB might fulfil an additional role in anaerobic electron transport. Mutants defective in dipZ (dsbD) were only slightly more sensitive to Cu⁺⁺ ions at concentrations above 5?mM than the parental strain, but strains defective in DsbA, DsbB, DsbC, DsbE or DsbF were unaffected. These results are consistent with our earlier proposals that DsbA, DsbB and DipZ (DsbD) are part of the same pathway for ensuring that haem groups are attached to the correct pairs of cysteine residues of apocytochromes c in the E. coli periplasm. However, neither DsbE nor DsbF are essential for the reduction of DipZ (DsbD).     

Menaquinone is an obligatory component of the chain catalyzing succinate respiration in Bacillus subtilis

The question was investigated as to whether the bacterial menaquinone (MK) is a component of the electron transport chain catalyzing succinate respiration in Bacillus subtilis. Three different methods were applied, and the following consistent results were obtained. (i) Solvent extraction of MK from the bacterial membrane caused total inhibition of the respiratory activities with succinate and NADH, while the activity of succinate dehydrogenase remained unaffected. The respiratory activities were restored onincorporation of vitamin K₁ into the membrane preparation. (ii) The membrane fraction of a B. subtilis mutant containing 15% of the wild-type amount of MK, respired succinate and NADH at reduced activities. Wild-type activities were restored on fusion of the preparation to liposomes containing vitamin K₁. (iii) The membrane fraction of B. subtilis catalyzed succinate oxidation by various water-soluble naphtho- or benzoquinones at specific activities exceeding to that of succinate respiration. The results suggest that MK is involved in succinate respiration, although its redox potential is unfavorable.Abbreviations MK menaquinone - MKH₂ reduced menaquinone - E₀' standard redox potential at pH 7 - PMS phenazine methosulfate - DCPIP 2,6-Dichlorophenol-indophenol - Q ubiquinone - Q₀ 2,3-dimethoxy-5-methyl-1,4-bezoquinone - DMN, 2,3 dimethyl-1,4-naphthoquinone - DMK demethylmenaquinone     

Evidence for the presence of di- and triphospho pyridine nucleotide dehydrogenase derivatives as consistent contaminants of purified beef heart cytochrome-c oxidase

Purified beef heart cytochrome-c oxidase preparations derived by three different laboratories contain NADH-K₃ Fe(CN)₆, NADH-nitrobluetetrazolium, and NADPH-nitrobluetetrazolium reductases. This is true of preparations exhibiting heme aa₃ to protein ratios considered indicative of an excellent purity. An apparent association of cytochrome-c oxidase and one or more of the contaminants persists through immunodiffusion and nondenaturing electrophoresis and, in addition, in one instance copurification of NADH-K₃ Fe(CN)₆ reductase and cytochrome-c oxidase to a constant ratio of specific activities was demonstrated. Cytochrome-c oxidase can be freed of the contaminants by equilibration with an NAD⁺-affinity matrix. As a concomitant of equilibration with the matrix, theK _M of cytochrome-c oxidase for ferrocytochrome-c is invariably decreased. Rate constants at low ferrocytochrome-c concentrations are consistently enhanced in all oxidase preparations upon equilibration with the NAD⁺ matrix. However, the effects of such equilibrations on the extrapolatedV _max varies from one preparation to another. Polyacrylamide gel electrophoresis in SDS-urea systems establishes that each of the preparations contains a minimum of three contaminants, each of an apparent formula weight of greater than 40,000 Daltons. NADH-NBT reductase was found to have a formula weight of approximately 46,000 Daltons. Their properties establish that NADH-K₃ Fe(CN)₆ and NADH-NBT reductases are separate proteins; the separate identity of NADPH-NBT reductase has not yet been determined.     

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.     

Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers

Changes in the level of the endoplasmicreticulum (ER) marker enzyme cytochrome-c reductase (EC 1.6.2.1) were followed with time of imbibition of de-embryonated half-seeds of barley (Hordeum vulgare L.) and the subsequent incubation of their aleurone layers in gibberellic acid (GA₃) and H₂O. During imbibition there is an increase in the level of cytochrome-c-reductase activity and in the amount of 280-nm absorbance associated with this enzyme. When aleurone layers are incubated for a further 42 h in water, there is a doubling of the cytochrome-c-reductase activity. In GA₃, the activity of cytochrome-c reductase reaches a maximum at 24 h of incubation and thereafter falls to below 70% of its level at the beginning of the incubation period. Changes in the cytochrome-c-reductase activity correlate with changes in the fine structure of the aleurone cell. The ER isolated in low Mg²⁺ from aleurone layers incubated in buffer for up to 18 h has buoyant density of 1.13–1.14 g cc^-1 while that from layers incubated in GA₃ for 7.5–18 h has a density of 1.11–1.12 g cc^-1. The -amylase (EC3.2.1.1) isolated with the organelle fraction by Sepharose gel filtration is associated with the ER on isopycnic and rate-zonal density gradients, and its activity can be enhanced by Triton X-100. The soluble -amylase fraction from Separose-4B columns, on the other hand, is not Triton-activated but is acid-labile. Acid phosphatase (EC3.1.3.2) is distributed in at least three peaks on isopycnic gradients. In low Mg²⁺ the second peak of activity has a density of 1.12 g cc^-1 in GA₃-treated tissue and 1.13–1.14 g cc^-1 in H₂O-treated tissue. With high-Mg²⁺ buffers, this peak of phosphatase activity disappears. Acid-phosphatase activity is not enhanced by Triton X-100 nor is it acid-labile.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid     

Molecular identification of the ten subunits of cytochrome-c reductase from potato mitochondria

Cytochrome-c reductase (EC 1.10.2.2.) from Solanum tuberosum L. comprises ten subunits with apparent molecular sizes of 55, 53, 51, 35, 33, 25, 14, 12, 11 and 10 kDa on 14% SDS-PAGE. The identity of the subunits was analysed by direct amino-acid sequencing via cyclic Edman degradation. A large-scale purification procedure for the enzyme complex based on affinity chromatography and gelfiltraton is described. All subunits were enzymatically fragmented and the generated peptides were separated by reverse-phase HPLC. Complete or partial sequence determination of 33 peptides comprising a total of nearly 500 amino acids showed, that cytochrome-c reductase from potato contains three respiratory proteins (cytochrome b, cytochrome c ₁ and the Rieske iron-sulfur protein), four small proteins with molecular sizes below 15 kDa (so-called Q-binding, hinge, cytochrome-c ₁-linked and core-linked proteins) and three proteins in the 50-kDa range which show similarity to members of the core/PEP/MPP protein family (core/processing enhancing protein/mitochondrial processing peptidase). In fact these subunits show highest sequence identity either to MPP or PEP, which is in line with earlier findings, that isolated cytochrome-c reductase from potato exhibits processing activity towards mitochondrial precursor proteins.Abbreviations MPP mitochondrial processing peptidase - PEP processing enhancing protein This research was supported by the Deutsche Forschungsgemeinschaft.     

Anaerobic induction of trimethylamine N-oxide reductase and cytochromes by dimethyl sulfoxide inEscherichia coli

Reduction of trimethylamine N-oxide is catalyzed by at least two enzymes inEscherichia coli: trimethylamine N-oxide reductase, which is anaerobically induced by trimethylamine N-oxide, and the constitutive enzyme dimethyl sulfoxide reductase. In this study, an increase in the specific activity of trimethylamine N-oxide reduction was observed in the anaerobic culture with dimethyl sulfoxide, but the specific activity of dimethyl sulfoxide reduction was not changed. The inducible enzyme trimethylamine N-oxide reductase was found in this culture. A marked expression of the structural genetorA for trimethylamine N-oxide reductase was also observed in atorA-lacZ gene fusion strain under anaerobic conditions with either trimethylamine N-oxide or dimethyl sulfoxide.l-Methionine sulfoxide and the N-oxides of adenosine, picolines, and nicotinamide slightly repressed expression of the gene. Membrane-boundb- andc-type cytochromes involved in the trimethylamine N-oxide reduction were also produced in a wild-type strain grown anaerobically with dimethyl sulfoxide. But thec-type cytochrome was not produced in thetorA-lacZ strain grown anaerobically with trimethylamine N-oxide or dimethyl sulfoxide; this suggests that there is a correlation between the expression oftorA and the synthesis of the cytochrome.