Aerobic and anaerobic respiratory systems in Campylobacter fetus subsp. jejuni grown in atmospheres containing hydrogen.

Maximum growth of Campylobacter fetus subsp. jejuni, strain C-61, occurred when the cultures were incubated with shaking in atmospheres containing approximately 30% hydrogen, 5% oxygen, and 10% CO2. Suspensions of cells grown under these conditions consumed oxygen with formate as the substrate in the presence of 0.33 mM cyanide, which completely inhibited respiration with ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine and with lactate. Spectroscopic evidence with intact cells suggested that a form of cytochrome c, reducible with formate but not with lactate or ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine, can be reoxidized by a cyanide-insensitive system. Analysis of membranes from the cells showed high- and low-potential forms of cytochrome c, cytochrome b, and various enzymes, including hydrogenase, formate dehydrogenase, and fumarate reductase. The predominant carbon monoxide-binding pigment appeared to be a form of cytochrome c, but the spectra also showed evidence of cytochrome o. The membrane cytochromes were reduced by hydrogen in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide at concentrations which prevented the reduction of cytochrome c with succinate as the electron donor. Reoxidation of the substrate-reduced cytochromes by oxygen was apparently mediated by cyanide-sensitive and cyanide-insensitive systems. The membranes also had hydrogen-fumarate oxidoreductase activity mediated by cytochrome b. We conclude that C. fetus jejuni has high- and low-potential forms of cytochrome which are associated with a complex terminal oxidase system.     

The electron transport system of the anaerobic Propionibacterium shermanii: cytochrome and inhibitor studies.

1. Electron transport particles obtained from cell-free extracts of Propionibacterium shermanii by centrifugation at 105000 times g for 3 hrs oxidized NADH, D,L-lactate, L-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too. 2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome alpha1 and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80--90%. 3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or "site I region", in the electron transport system of P. shermanii. 4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor and reached 80--100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase. 5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions. 6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations of the inhibitor. Inhibitors of terminal oxidases were inactive, except for carbon monoxide.     

Four different b-type cytochromes in the halophilic archaebacterium, Halobacterium halobium

The halophilic archaebacterium, Halobacterium halobium has been found to contain four different b-type cytochromes. The four components were recognized by their potentiometric characteristics in situ in their functional environment in the membrane of H. halobium. Oxidation-reduction midpoint potentials of these four b-type cytochromes were determined to be +261, +160, +30, and -153 mV, respectively. We also demonstrate that the pathway involved in the transport of reducing equivalents from succinate to oxygen proceeds through the b-type cytochromes with oxidation-reduction midpoint potentials of +261 and +161 mV. The cytochrome with oxidation-reduction midpoint potential of -153 mV was not substrate reducible by NADH but was chemically reducible by dithionite. Antimycin inhibits reduction of b-type cytochrome in the succinate pathway, but has no effect on b-type cytochrome reduction when reducing equivalents are provided by NADH. The carbon monoxide difference spectrum of H. halobium membranes shows at least one carbon monoxide-binding b-type cytochrome, indicating a terminal oxidase. A scheme for electron transport in H.halobium involving the b-type cytochromes and terminal oxidase is suggested.     

Respiratory systems and cytochromes in Campylobacter fetus subsp. intestinalis.

Cell suspensions of Campylobacter fetus subsp. intestinalis grown microaerophilically in complex media consumed oxygen in the presence of formate, succinate, and DL-lactate, and membranes had the corresponding dehydrogenase activities. The cells and membranes also had ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity which was cyanide sensitive. The fumarate reductase activity in the membranes was inhibited by p-chloromercuriphenylsulfonate, and this enzyme was probably responsible for the succinate dehydrogenase activity. Cytochrome c was predominant in the membranes, and a major proportion of this pigment exhibited a carbon monoxide-binding spectrum. Approximately 60% of the total membrane cytochrome c, measured with dithionite as the reductant, was also reduced by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. A similar proportion of the membrane cytochrome c was reduced by succinate under anaerobic conditions, whereas formate reduced more than 90% of the total cytochrome under these conditions. 2-Heptyl-4-hydroxyquinoline-N-oxide inhibited reduction of cytochrome c with succinate, and the reduced spectrum of cytochrome b became evident. The inhibitor delayed reduction of cytochrome c with formate, but the final level of reduction was unaffected. We conclude that the respiratory chain includes low- and high-potential forms of cytochromes c and b; the carbon monoxide-binding form of cytochrome c might function as a terminal oxidase.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

The respiratory system of Chromobacterium violaceum grown under conditions of high and low cyanide evolution.

The particulate fraction of disrupted Chromobacterium violaceum grown under cyanide-evolving conditions was unable to oxidize ascorbate plus N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD), but oxidized NADH and succinate by a linear respiratory pathway which was very resistant to inhibition by cyanide. When the bacteria were grown under conditions where little cyanide evolution occurred, particulate fractions developed the ability to oxidize ascorbate-TMPD by a pathway highly sensitive to cyanide inhibition; respiratory activity with NADH and succinate proceeded via both the cyanide-sensitive and-resistant pathways. Studies with respiratory inhibitors, and the cytochrome compositions of the fractions derived from cultures grown under both conditions, are presented. A soluble, carbon monoxide-binding cytochrome c was found, and this appears similar to those found recently in Beneckea natiegens, methylotrophic bacteria and the marine pseudomonad B16.     

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.     

Electron transport in the membrane of lutoids from the latex of Hevea brasiliensis.

1. An antimycin-insensitive NADH-cytochrome c oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex of Hevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH. 2. Two beta-type cytochromes are present in the membranes. Cytochrome beta563 is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 degrees K. A second cytochrome, cytochrome beta561, seems to be reducible by hydrosulfite only. 3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochrome P-450 could not be demonstrated. 4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.     

NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane.

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.     

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.     

Characterization of cyanide-insensitive respiration in mitochondria and submitochondrial particles of Moniliella tomentosa

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O(2) as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O(2) concentration for saturation than does cytochrome c oxidase. 4. The apparent K(m) for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4-10 times higher when measured in submitochondrial particles. The apparent K(m) for NADH is roughly the same for both oxidases. 5. The apparent K(m) values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent K(m) for AMP, acting as a stimulator of the alternative oxidase, is the same (25mum) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.     

Cytochrome spectrum of an obligate anaerobe, Eubacterium lentum.

An obligately anaerobic bacterium, Eubacterium lentum, was shown to contain cytochromes a, b, and c and a carbon monoxide-binding pigment. Extracts of cells grown with hemin gave a typical absorption spectrum for cytochrome c with maxima at 424, 525, and 553 nm. Extracts from cells grown in the absence of hemin also had an absorption peak corresponding to cytochrome b (562 nm) in their reduced versus oxidized spectrum. Extraction of hemes and formation of pyridine hemochromes allowed quantitation of protoheme IX and heme c. Large amounts of cytochrome c masked the presence of cytochrome b in cells grown in medium containing hemin. When cells were grown in the presence of 50 mM nitrate, cytochrome A (606 nm) was detected. In anaerobic extracts of cells grown either with or without nitrate, cytochromes b and c were reduced by formate and oxidized by NO3. Cytochrome a appeared to be partially oxidized by NO3 and completely oxidized by air.     

The inhibition of plant mitochondrial respiration by the synthetic analog of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT)

The quinone analog, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), has been shown to inhibit cyanide-sensitive and cyanide-insensitive respiration in higher plant mitochondria. The inhibition is dependent upon the concentration of mitochondrial protein. The low concentrations of UHDBT required to inhibit the cyanide-sensitive pathway (microM) and the cyanide-insensitive pathway (nM) indicate that UHDBT is acting as a tight-binding inhibitor of ubiquinol oxidation. Inhibition of both pathways was dependent upon pH. It is shown that UHDBT appears to be a less potent inhibitor of cyanide-sensitive NADH oxidation than of cyanide-sensitive succinate oxidation, and that the pH dependence of inhibition of these two pathways differs. The inhibition of NADH and succinate oxidation by the cyanide-insensitive pathway shows similar pH dependences although at a given pH NADH oxidation is more susceptible to inhibition than succinate oxidation.     

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.     

NADPH Oxidase System as a Superoxide-generating Cyanide-Resistant Pathway in the Respiratory Chain of Corynebacterium glutamicum

The respiratory chain of Corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. The membranes of C. glutamicum had NADH, succinate, lactate, and NADPH oxidase activities, and menaquinone, and cytochromes a ₅₉₈, b _562(558), and c ₅₅₀ as respiratory components. The NADH, succinate, lactate, and NADPH oxidase systems, all of which were more cyanide-resistant than N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity (cytochrome aa ₃ terminal oxidase), had different sensitivities to cyanide; the cyanide sensitivity of these oxidase systems increased in the order, NADPH, lactate, NADH, and succinate. Taken together with the analysis of redox kinetics in the cytochromes and the effects of respiratory inhibitors, the results suggested that there is a cyanide-resistant bypass oxidase branching at the menaquinone site, besides cyanide-sensitive cytochrome oxidase in the respiratory chain. H⁺/O measurements with resting cells suggested that the cyanide-sensitive respiratory chain has two or three coupling sites, of which one is in NADH dehydrogenase and the others between menaquinone and cytochrome oxidase, but the cyanide-resistant bypass oxidase may not have any proton coupling site. NADPH and lactate oxidase systems were more resistant to UV irradiation than other systems and the UV insensitivity was highest in the NADPH oxidase system, suggesting that a specific quinone resistant to UV or no such a quinone works in at least NADPH oxidase system while the UV-sensitive menaquinone pool does in other oxidase systems. Furthermore, superoxide was generated in well-washed membranes, most strongly in the NADPH oxidase system. Thus, it was suggested that the cyanide-resistant bypass oxidase system of C. glutamicum is related to the NADPH oxidase system, which may be involved in generation of superoxide anions and probably functions together with superoxide dismutase and catalase.     

Effect of oxygen supply during growth on the production of cytochromes, enzymes, and acid end products by Haemophilus parasuis.

Haemophilus parasuis, grown under conditions of high aeration, was found to lack a tricarboxylic acid cycle but to possess phosphoenolpyruvate carboxylase and a reductive pathway leading to the production of succinate. Such organisms contained approximately equal quantities of b-, c-, and d-type cytochromes and excreted acetate. When the oxygen supply for growth was either reduced or eliminated, the specific activities of phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, and NADH: fumarate oxidoreductase were increased substantially, and the acid products were succinate, acetate, and formate. Organisms grown under the latter conditions also contained increased quantities of b- and c-type cytochromes, some of which were low-potential cytochromes. These low-potential cytochromes were reduced by NADH and oxidized by fumarate, and hence, appeared to be components of NADH: furmarate oxidoreductase. Our results indicate that in H. parasuis, growing aerobically in medium containing glucose, the sole function of the reductive pathway is to provide intermediates for biosynthetic processes, and oxygen is the preferred electron acceptor. As the supply of oxygen is reduced or eliminated, the reductive pathway becomes more involved in NAD+ recycling and fumarate becomes the acceptor. In effect, irrespective of the oxygen supply, the growth of H. parasuis is absolutely dependent upon the presence of an electron transport system.     

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.     

Metabolism of Pipecolic Acid in a Pseudomonas Species IV. Electron Transport Particle of Pseudomonas putida

Baginsky, Marietta L. (University of California, San Francisco Medical Center, San Francisco), and Victor W. Rodwell. Metabolism of pipecolic acid in a Pseudomonas species. IV. Electron transport particle of Pseudomonas putida. J. Bacteriol. 92:424-432. 1966.-Enzymes of Pseudomonas putida P2 catalyzing oxidation of pipecolate to Delta(1)-piperideine-6-carboxylate are located in a subcellular fraction sedimenting at 105,000 x g. Since this fraction resembles the mammalian electron transport particle in both chemical composition and enzymatic activities, it was termed Pseudomonas P2 electron transport particle (P2-ETP). P2-ETP contains flavin adenine dinucleotide, flavin mononucleotide, iron, copper, and both b- and c-type cytochromes. The reduced type b cytochrome has absorption maxima at 558 to 559, 530, and 427 mmu. Its oxidized pyridine hemochromogen has an absorption maximum at 406 mmu, with a shoulder at 564 mmu. On dithionite reduction, absorption bands with maxima at 556, 522, and 418 mmu are obtained. The reduced type c cytochrome has absorption maxima at 552, 520, and 422 mmu; its reduced pyridine hemochromogen has maxima at 551, 516 to 519, and 418 mmu. No type a cytochrome was detected. P2-ETP catalyzes oxidation of pipecolate and of reduced nicotinamide adenine dinucleotide (NADH(2)) by oxygen. It can also oxidize these compounds, as well as succinate and reduced nicotinamide adenine dinucleotide phosphate, with 2,6-dichlorophenol-indophenol as electron acceptor. Mammalian cytochrome c can be used as an alternate artificial electron acceptor for the oxidation of pipecolate and succinate, but not for oxidation of NADH(2).     

The electron transport system of the anaerobic Propionibacterium shermanii

1. Electron transport particles obtained from cellfree extracts of Propionibacterium shermanii by centrifugation at 105000xg for 3 hrs oxidized NADH, d,l-lactate, l-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too.
2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome a ₁ and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80–90.
3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or “site I region”, in the electron transport system of P. shermanii.
4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor, and reached 80–100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase.
5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions.
6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations of the inhibitor. Inhibitors of terminal oxidases were inactive, except for carbon monoxide.

     

Pyridine nucleotides and H2 as electron donors to the respiratory and photosynthetic electron-transfer chains and to nitrogenase in Anabaena heterocysts

The pathways through which NADPH, NADH and H₂ provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O₂ uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H₂ supported cyanide-sensitive, respiratory O₂ uptake and light-enhanced, cyanide-insensitive uptake of O₂ resulting from electron donation to O₂ at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O₂ or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H₂ supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H₂ as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented.