Respiratory physiology and cytochrome content ofLegionella pneumophila

The cytochrome composition of membrane vesicles ofLegionella pneumophila has been examined by low temperature (77°K) and room temperature difference spectroscopy, and cytochromes of thec, b, a, andd types have been detected. The presence ofc-type cytochrome was verified by formation of the pyridine ferrohemochromogen. A carbon monoxide-bindingc-type cytochrome was detected in CO-reduced minus reduced difference spectra and may also function in cytochromec reductase activity. Respiratory activities were determined for membrane vesicles, and reduced nicotinamide adenine dinucleotide (NADH) was the most rapidly oxidized substrate (199 nmol per min per mg protein), followed by succinate and malate. Cytochrome oxidase activity was demonstrated using ascorbate andN,N,N,N-tetramethyl-p-phenylenediamine (TMPD) (39 nmol per min per mg of protein). High levels of cyanide (K _i =10 mM) inhibited NADH oxidation, while low levels of 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO, 18 and 37 M) inhibited NADH oxidation by nearly 90%. The respiratory chain appeared to be complex and terminated by at least three terminal oxidases. Superoxide dismutase activity, but not catalase activity, was detected in cellular extracts.     

Electron transport chain of Zymomonas mobilis

The interaction of the membrane-bound glucose dehydrogenase from the anaerobic but aerotolerant bacterium Zymomonas mobilis with components of the electron transport chain has been studied. Cytoplasmic membranes showed reduction of oxygen to water with the substrates glucose or NADH. The effects of the respiratory chain inhibitors piericidin, capsaicin, rotenone, antimycin, myxothiazol, HQNO, and stigmatellin on the oxygen comsumption rates in the presence of NADH or glucose as substrates indicated that a complete and in the most parts identical respiratory chain is participating in the glucose as well as in the NADH oxidation. Furthermore, the presence of coenzyme Q₁₀ (ubiquinone 10) in Z. mobilis was demonstrated. Extraction from and reincorporation of the quinone into the membranes revealed that ubiquinone is essential for the respiratory activity with glucose and NADH. In addition, a membrane-associated tetramethyl-p-phenylene-diamine-oxidase activity could be detected in Z. mobilis.Abbreviations ABTS 2,2-Azino-di-[3-ethyl-benzthiazolinesulfonate (6)] - GDH glucose dehydrogenase - HQNO 2-heptyl-4-hydroxy-quinoline-N-oxide - PQQ pyrroloquinoline quinone - TMPD N,N,N,N-tetramethyl-p-phenylene-diamine     

The role ofc-type cytochromes in the terminal respiratory chain of the methylotrophic bacteriumMethylophilus methylotrophus

Whole cells of the methylotrophic bacteriumMethylophilus methylotrophus cultured under methanol-limited conditions contain approximately equal amounts of two majorc-type cytochromes,c _H andc _L. Virtually all of the cytochromec _H, and over one-third of the cytochromec _L, are loosely attached to the periplasmic surface of the respiratory membrane whence they can be released by sonication or by washing cells in ethylenediaminetetraacetate (EDTA). The latter causes inhibition of methanol oxidase activity and stimulation of ascorbate-N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) oxidase activity, neither of which effects are reversible by divalent metal ions. Kinetic analyses indicate that ascorbate-TMPD is oxidised via two routes, viz. a slow low-affinity pathway involving loosely membrane-boundc-type cytochromes plus cytochrome oxidaseaa ₃, and a faster higher-affinity pathway involving the firmly membrane-bound cytochrome oxidasec _L o complex; the former route predominates in the presence of divalent metal ions, and the latter route after exposure to EDTA. These and other results are discussed in terms of the spatial organisation of the terminal respiratory chain, and of the role ofc-type cytochromes in the oxidation of methanol and ascorbate-TMPD.Abbreviations EDTA Enthylenediaminetetraacetate - PMS Phenazinemethosulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - SDS Sodium dodecylsulphate - I₅₀ Concentration of inhibitor required to give 50% inhibition of enzyme activity - PQQ Pyrroloquinoline quinone     

Electron transport-linked proton translocation at nitrite reduction inCampylobacter sputorum subspeciesbubulus

Campylobacter sputorum subspeciesbubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate andL-lactate are used as hydrogen donors. Cells ofC. sputorum grown with nitrate or nitrite contain cytochromes of theb-andc-type and a carbon monoxide-binding cytochromec. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate orL-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenyl-hydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. H⁺/O values and H⁺/NO ₂ ^- values were measured with ascorbate + N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) andL-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a H⁺/2e value of 2.0. The H⁺/O and H⁺/NO ₂ ^- values predicted by this scheme are in good agreement with the experimental values.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - MTPP⁺ methyltriphenylphosphonium cation - TMPD N,N,N,N-tetramethyl-p-phenylenediamine; H⁺/O (H⁺/NO ₂ ^- ), number of protons liberated in the outer bulk phase at the reduction of one atom O (one ion NO ₂ ^- ); H⁺/2e (q⁺/2e), number of protons (charges) translocated across the cytoplasmic membrane during flow of two electrons to an acceptor     

Inhibition of nitrate reduction by light and oxygen in Rhodobacter sphaeroides forma sp. denitrificans

Light inhibited each step of the denitrification process in whole cells of Rhodobacter sphaeroides forma sp. denitrificans. This inhibition by light was prevented in the presence of exogenous electron donors like N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) plus ascorbate or in the presence of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone). Addition of myxothiazol restored the inhibition by light in uncoupled cells. Measurements of light-induced absorbance changes under these conditions showed that this inhibition is due, for the steps of reduction of nitrite to dinitrogen, to the photooxidation of cytochromes c ₁ plus c ₂ and not due to the photoinduced membrane potential. Moreover, the presence of oxygen inhibited almost all of the reduction of nitrate and nitrous oxide but only 70% of the reduction of nitrite to nitrous oxide. These inhibitions were overcome in the presence of TMPD plus ascorbate. This implies that the inhibition in presence of oxygen was due to a diversion of the reducing power from the denitrifying chain to the respiratory chain. It was concluded from this series of experiments that the reduction of nitrate to nitrite is inhibited when the ubiquinone pool is partly oxidized and that nitrite and nitrous oxide reductions are inhibited when cytochromes c ₁ plus c ₂ are oxidized by photosynthesis or respiration.Abbreviations R Rhodobacter - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - HOQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - CCCP carbonyl cyanide m-chlorophenylhydrazone - cytochrome c ₁ cytochrome c ₂ plus cytochrome c ₁     

Photooxidase activity of isolated chromatophores and intact cells of phototrophic bacteria

Illumination causes an uptake of oxygen by isolated chromatophores of purple and green bacteria incubated with electron donors. Photooxidase activity of Rhodospirillum rubrum, Chromatium minutissimum, Rhodopseudomonas sphaeroides and Thiocapsa roseopersicina chromatophores is sensitive, and photooxidase activity of Ectothiorhodospira shaposhnikovii and Chlorobium limicola f. thiosulfatophilum is resistant to o-phenanthroline. O₂ uptake by illuminated chromatophores of R. rubrum and C. limicola is stimulated upon the increase of pH of incubation mixture from 5 to 9. Photooxidase activity is also manifested in the intact bacterial cells and not merely in the isolated chromatophores. O₂ uptake by the illuminated R. rubrum cells treated with CN^- is stimulated by 2-heptyl-4-hydroxyquinoline-N-oxide and a protonophorous uncoupler. The interaction of the photosynthetic and respiratory systems of the electron transfer in the bacterial cells and the probable causes of the strong anaerobic way of life of the green sulfur bacteria are discussed.HQNO 2-heptyl-4-hydroxyquinoline-N-oxide - TMPD N,N,-N,N-tetramethyl-p-phenylenediamine     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

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     

Properties of the menaquinol oxidase (Qox) and of qox deletion mutants of Bacillus subtilis

Menaquinol oxidase isolated from the membrane of Bacillus subtilis W23 was found to consist of four polypeptides (QoxA, B, C, and D) that were predicted by the sequence of the qox operon of B. subtilis 168 (Santana et al. 1992). The preparation contained 7 mol cytochrome aa ₃ per g protein, which corresponds to 2mol heme A per mol enzyme of 144 kDa molecular mass. Respiration with dimethylnaphthoquinol catalyzed by the enzyme was ten times faster than that with menadiol. Activities with more electropositive quinols were negligible. The activity of the enzyme was inhibited by equimolar amounts of HQNO, while antimycin, myxothiazol, and stigmatellin were more than tenfold less effective. When cells of both strains of B. subtilis (W23 and 168) were grown with glucose, quinol respiration was an order of magnitude more active than respiration with N,N,N,N-tetramethyl-1,4-phenylenediamine plus ascorbate. Surprisingly, the same result was obtained with mutant strains lacking qoxB. As cytochromes a and d were virtually absent, a second quinol oxidase, possibly of the cytochrome o-type, was apparently formed by the mutants.Abbreviations cat Chloramphenicol resistance gene - cta Cytochrome oxidase genes - DMN 2,3-Dimethyl-1,4-naphthoquinone - DMNH ₂ Reduced DMN - HQNO 2-(n-Heptyl)-4-hydroxyquinoline-N-oxide - qox Quinol oxidase genes - TMPD N,N,N,N-tetramethyl-1,4-phenylenediamine     

Evidence for a dual role of cytochrome c-553 and plastocyanin in photosynthesis and respiration of the cyanobacterium,Anabaena variabilis

The cytochrome oxidase activity (oxygen uptake in the dark) of a membrane preparation from Anabaena variabilis was found to be stimulated by cytochrome c-553 and plastocyanin obtained from this alga. Cytochrome c from horse heart was as active as cytochrome c-553, whereas little or no stimulation of oxygen uptake was obtained with cytochromes c ₂ from two Rhodospirillaceae, the plastidic cytochrome c-552 from Euglena, and plastocyanin from spinach. Cytochrome c-553 (A. variabilis) stimulated photosystem 1 activity in the same preparation much more than cytochrome c (horse heart). The results indicate that cytochrome c-553 and plastocyanin, besides their established function as electron donors of photosystem 1, participate in respiratory electron transport as reductants of a terminal oxidase. Photooxidation and dark oxidation show a different donor specificity.Abbreviations Chl chlorophyll a - TMPD N,N,N,N-tetramethyl-p-phenylenediamine     

Roles of respiratory oxidases in protecting Escherichia coli K12 from oxidative stress

Isogenic strains of Escherichia coli that were defective in either of the two major aerobic terminal respiratory oxidases (cytochromes bo and bd) or in the putative third oxidase (cytochrome bd-II) were studied to elucidate role(s) for oxidases in protecting cells from oxidative stress in the form of H₂O₂ and paraquat. Exponential phase cultures of all three oxidase mutants exhibited a greater decline in cell viability when exposed to H₂O₂ stress compared to the isogenic parent wild-type strain. Cytochrome bo mutants showed the greatest sensitivity to H₂O₂ under all conditions studied indicating that this oxidase was crucial for protection from H₂O₂ in E. coli. Cell killing of all oxidase mutants by H₂O₂ was by an uncharacterized mechanism (mode 2 killing) with cell growth rate affected. The expression of (katG-lacZ), an indicator of intracellular H₂O₂, was 2-fold higher in a cydAB::kan mutant compared to the wild-type strain at low H₂O₂ concentrations (< 100 M) suggesting that cytochrome bd mutants were experiencing higher intracellular levels of H₂O₂. Protein fusions to the three oxidase genes demonstrated that expression of genes encoding cytochrome bd, but not cytochrome bo or cytochrome bd-II was increased in the presence of external H₂O₂. This increase in expression of (cydA-lacZ) by H₂O₂ was further enhanced in a cyo::kan mutant. The level of cytochrome bd determined spectrally and (cydA-lacZ) expression was 5-fold and 2-fold higher respectively in an rpoS mutant compared to isogenic wild-type cells suggesting that RpoS was a negative regulator of cytochrome bd. Whether the effect of RpoS is direct or indirect remains to be determined.     

The cyclic electron pathways around photosystem I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage

The photoacoustic technique was used to measure energy storage by cyclic electron transfer around photosystem I in intact Chlamydomonas reinhardtii cells illuminated with far-red light (>715 nm). The in-vivo cyclic pathway was characterized by investigating the effects of various chemicals on energy storage. Participation of plastoquinone and ferredoxin in the cyclic electron flow was confirmed by the complete suppression of energy storage in the presence of the plastoquinol antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the ferredoxin inhibitors/competitors methylviologen, phenylmercuric acetate and p-benzoquinone. Two alternative electron cycles are demonstrated to operate in vivo. One cycle is sensitive to antimycin A, myxothiazol and 2-(n-heptyl)-4-hydroxyquinoline N-oxide (HQNO) and is catalyzed by ferredoxin which reduces plastoquinone through a route involving cytochrome b ₆ and its protonmotive Q-cycle. The other cycle is unaffected by the above-mentioned inhibitors but is sensitive to N-ethylmaleimide (NEM), an inhibitor of the ferredoxin-NADP reductase, and 2-monophosphoadenosine-5-diphosphoribose (PADR), an analogue of NADP, showing that the electron recycling was mediated by NADPH. Possibly, electrons enter the plastoquinone pool through the action of a NAD(P)H dehydrogenase, which is insensitive to classical inhibitors of the mitochondrial NADH dehydrogenase. Loss of energy storage by photosystem-I-driven cyclic electron transfer in farred light was observed only when antimycin A, myxothiazol or HQNO was used in combination with NEM or PADR. Analysis of the light-intensity dependence and the rate of in-vivo cyclic electron transfer in the presence of various inhibitors indicates that the NADPH-dependent electron-cycle is the preferential cyclic pathway in Chlamydomonas cells illuminated with far-red light.Abbreviations A^max maximal photothermal signal - Cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ES photochemical energy storage - FNR ferredoxin NADP⁺ reductase - HQNO 2-(n-heptyl)-4-hydroxyquinoline N-oxide - NEM N-ethylmaleimide - P₇₀₀ reaction-center pigment of PSI - PADR 2-monophosphoadenosine-5-diphosphoribose - pBQ p-benzoquinone - PMA phenylmercuric acetate We are very grateful to Dr. M.-H. Montane (Cadarache, Saint-Paul-lez-Durance, France) for her advice in the electroporation experiments.     

Structure and function of the yeast vacuolar membrane proton ATPase

Our current work on a vacuolar membrane proton ATPase in the yeastSaccharomyces cerevisiae has revealed that it is a third type of H⁺-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F₀F₁-type ATP synthase and the plasma membrane E₁E₂-type H⁺-ATPase. The vacuolar membrane H⁺-ATPase is composed of three major subunits, subunita (M _r =67 kDa),b (57kDa), andc (20 kDa). Subunita is the catalytic site and subunitc functions as a channel for proton translocation in the enzyme complex. The function of subunitb has not yet been identified. The functional molecular masses of the H⁺-ATPase under two kinetic conditions have been determined to be 0.9–1.1×10⁵ daltons for single-cycle hydrolysis of ATP and 4.1–5.3×10⁵ daltons for multicycle hydrolysis of ATP, respectively.N,N-Dicyclohexylcarbodiimide does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazolesensitive catalytic site. Cloning of structural genes for the three subunits of the H⁺-ATPase (VMA1, VMA2, andVMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.Abbreviations CCCP carbonyl cyanidem-chlorophenyl hydrazone - DCCD N,N-dicyclohexylcarbondiimide - DES diethylstilbestrol - DIDS 4,4-diisothiocyano-2,2-stilbene disulfonic acid - NBD-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Pi inorganic phosphate - SDS sodium dodecylsulfate - SF6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile - SITS 4-acetamide-4-isothiocyanatostilbene-2,2-disulfonic acid - ZW3-14 N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate     

Electron transport reactions in respiratory particles of hydrogenase-induced Anacystis nidulans

Respiratory particles from hydrogen-grown Anacystis nidulans were found to oxidize H₂, NADPH, NADH, succinate and ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine at rates corresponding to 28, 15, 6, 2.5, and 70 nmol O₂ taken up x mg protein^–1xmin^–1, respectively. The particles were isolated by brief sonication of lysozyme-pretreated cells. Respiratory activities were studied in terms of both substrate oxidation and O₂ uptake. The stoichiometry between oxidation of H₂, NADPH, NADH or succinate, and consumption of O₂ was calculated to be 1.95+-0.1 with each substrate.Inhibitors of flavoproteins did not affect the oxyhydrogen reaction while 2-n-heptyl-8-hydroxyquinoline-N-oxide as well as compounds known to block the terminal oxidase impaired the oxidation of both H₂ and of NAD(P)H or succinate in a parallel fashion. No additivity of O₂ uptake was observed when NADPH, NADH or succinate was present in addition to H₂. Instead, H₂ uptake was depressed under such conditions, and also the oxidation of NAD(P)H or succinate was increasingly lowered by increasing H₂ tensions.The results suggest that in Anacystis molecular hydrogen is oxidized through the same type of respiratory chain as are NAD(P)H and succinate. Moreover, the cyanide-resistant branch of respiratory O₂ uptake will be discussed, and a few results obtained with particles prepared from thylakoid-free Anacystis will also be presented.Abbreviations BAL 2,3-dimercaptopropanol-(1) - DCPIP 2,6-dichlorophenolindophenol - HOQNO 2-n-heptyl-8-hydroxyquinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethane - TTFA thenoyltrifluoroacetone NAD(P)H indicates NADPH and/or NADH     

Membrane-bound NADPH dehydrogenase- and ferredoxin: NADP oxidoreductase activity involved in electron transport during acetate oxidation to CO2 in Desulfobacter postgatei

Desulfobacter postgatei grows on acetate and sulfate as energy source. The oxidation of acetate to 2 CO₂ proceeds via the citric acid cycle involving membrane-bound succinate dehydrogenase and membrane-bound malate dehydrogenase. We report here that the organism contains membrane-bound NADPH dehydrogenase and ferredoxin: NADP oxidoreductase for the reoxidation of NADPH and reduced ferredoxin generated during isocitrate- and 2-oxoglutarate oxidation, respectively. The presence of proton translocating ATPase activity is also described.NADPH dehydrogenase and succinate dehydrogenase were found to be electrically connected within the membrane and electron transfer between these two enzymes was shown to be coupled with proton translocation. The membrane fraction catalyzed the oxidation of NADPH with fumarate and the reduction of NADP with succinate. NADPH oxidation with fumarate was stimulated by protonophores and inhibited by the proton translocating ATPase inhibitor dicyclohexylcarbodiimide (DCCD) and by heptylhydroxyquinoline-N-oxide (HQNO); inhibition by DCCD was relieved by protonophores. NADP reduction with succinate was dependent on ATP and inhibited by protonophores, DCCD, and HQNO. The membrane fraction also mediated the oxidation of NADPH with the water soluble menaquinone analogue dimethylnaphthoquinone (DMN) and the reduction of fumarate with DMNH₂. Only the former reaction was stimulated by protonophores and only the latter reaction was inhibited by HQNO. This suggests that the NADPH dehydrogenase reaction is the site of energy conservation and the succinate dehydrogenase is the site of HQNO inhibition.Non-standard abbreviations APS Adenosine 5-phosphosulfate - DCCD N,N-dicyclohexylcarbodiimide - DCPIP 2,6-dichloroindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - HQNO 2(n-heptyl)-4-hydroxyquinoline-N-oxide - TCS 3,5,3,4-tetrachlorosalicylanilide - Tricine N-tris-(hydroxymethyl)methylglycine - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - SF-6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile     

Reductive activation of the methyl-tetrahydromethanopterin: coenzyme M methyltransferase from Methanobacterium thermoautotrophicum strain ΔH

The enzymatic conversion of formaldehyde to CH₃S-CoM in crude extracts of Methanobacterium thermoautotrophicum was used as a means to investigate the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction. All components necessary for formaldehyde conversion were shown to be present in a soluble protein fraction. This soluble cell fraction still contained a major amount of corrinoids. Apart from tetrahydromethanopterin no other soluble cofactors were required for formaldehyde conversion. The dependence of the system on catalytic amounts of ATP was shown to be specific. Several nucleoside triphosphates or ADP were unable to substitute for ATP. Remarkably, various strong reducing systems, especially titanium(III)citrate could replace ATP to a large extent. The ATP-dependent formaldehyde conversion to CH₃S-CoM was inhibited in the presence of nitrous oxide, detergents or 2,3-dialdehyde-ATP. The results support a role for a corrinoid protein in the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction at which ATP is involved in the activation of this protein, probably in the conversion of inactive B_12a or B_12r to active B_12s.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - BES 2-bromoethanesulfonate - BCE boiled cell-free extract - DTT dithiothreitol - TCS 3,3,4,5-tetrachlorosalicylanilide - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - AMP-PNP 5-adenylyl imidophosphate     

Electron transport and respiration in Beggiatoa and Vitreoscilla

Seven strains of bacteria belonging to the Beggiatoa-Vitreoscilla group were studied for their respiratory activity and for the presence of electron transport conponents. All strains tested oxidized [1-¹⁴C] and [2-¹⁴C] acetate to ¹⁴CO₂ at relatively high rates. All strains tested were N,N,N,N-tetramethylphenylenediamine (TMPD)-oxidase positive and contained spectra representing a-type and carbon monoxide-binding cytochromes. Most of the strains also contained spectra representing c-type and b-type cytochromes. Beggiatoa alba B18LD contained b-type, a-type, c-type and CO-binding cytochromes, the latter two being located in the 144,000 x g soluble fraction. B. alba also contained ubiquinone-8 as its only detectable quinone.Non-standard abbreviations BSS basal salts solution - BH Beggiatoa heterotrophic medium - BSO Beggiatoa sulfide oxidation medium - TMPD N,N,N,N-tetramethylphenylenediamine - Q8 ubiquinone-8     

Evidence for two functionally distinct hydrogenases in Anacystis nidulans

The effect of growth conditions on aerobic and anaerobic hydrogenase activities of Anacystis nidulans was studied. It was found that the two hydrogenase activities both of which were confined to the particulate fraction of cell-free extracts correlated in an opposite way with growth temperature: The algae were always grown photoautotrophically in presence of H₂ but after growth at 25° C a significant oxyhydrogen reaction contrasted with negligible photoreduction rates while the opposite was true after growth at 40°C. A similar correlation between incubation temperature and induction of the respective hydrogenase activity was also observed with resting cells.Kinetic analysis of the two different types of hydrogenase — catalysed reactions with Anacystis membranes yielded the following Michaelis-Mentenparameters: K _M=55 M H₂ and v _max=0.12 mol H₂ per min and mg protein for the oxyhydrogen reaction, and K _M=170 M H₂ and v _max=0.3 mol H₂ per min and mg protein for the photoreductions. Also the dependences of oxyhydrogen and of photoreduction activities on pH and on temperature were measured; both pH and temperature profiles were found to be markedly different for each type of H₂-supported reaction.The results are discussed as pointing to the possible occurrence of two functionally distinct hydrogenase enzymes which can be synthesized by Anacystis in response to the conditions of induction.Abbreviations BO p-benzoquinone - CAP chloramphenicol - chl chlorophyll - cytc horse heart cytochrome c - DCMU 3-(34-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - fd ferredoxin - FeCy ferricyanide - MB methylene blue - MV methyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethan     

The enzymatic system thiosulfate: Cytochrome c oxidoreductase from photolithoautotrophically grown Chromatium vinosum

Chromatium vinosum cells form a vesicular type intracytoplasmic membrane system during phototrophic growth on thiosulfate.—An enzyme protein transferring electrons from thiosulfate to cytochromes of type c was enriched from S-144. The colorless thiosulfate: cytochrome c oxidoreductase was characterized by a molecular weight of 36,000 (after dodecylsulfate treatment) and 35,000 (by gel filtration). Isoelectric focusing revealed a pI range of 4.4 to 4.7. Apparent K _m values for the cytochromes tested were in the M range. — The endogenous electron acceptor compound, isolated from the chromatophore fraction P-144, was found to be a membrane-bound cytochrome c-552. The homogeneous cytochrome protein had an average pI value of 4.65 and a molecular weight of 71,500 determined by gel filtration. By dodecylsulfate electrophoresis it was cleaved into two proteins representing particle weights of 45,000 and 20,000.Abbreviations HiPIP high potential nonheme iron protein - IEF isoelectric focusing - SDS dodecylsulfate, sodium salt - Temed N,N,N,N-tetramethylethylenediamine     

The functional localization of cytochromes b in the respiratory chain of anaerobically grown Proteus mirabilis

The functional localization of the cytochromes b found in anaerobically grown Proteus mirabilis was investigated. From light absorption spectra, scanned during uninhibited and HQNO-inhibited electron transport to various electron acceptors, it was concluded that all cytochromes b function between two HQNO inhibition sites, or more probably in a Q- or b-cycle.Abbreviation HQNO= 2-n-heptyl-4-hydroxy-quinoline-N-oxide