Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium.
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Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when Clostridium cellulolyticum was cultured on medium containing cellobiose and yeast extract. Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD+ ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed. These data indicate a better control of the carbon flow on mineral salts medium than on complex medium. By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate. At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation. A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production. In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates. Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow. The same maximum growth rate (0.150 h-1) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization. On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH. Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase. (+info)
Simultaneous presence of p47(phox) and flavocytochrome b-245 are required for the activation of NADPH oxidase by anionic amphiphiles. Evidence for an intermediate state of oxidase activation.
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We have examined the kinetics of NADPH oxidase activation induced by arachidonic acid or SDS in a cell-free system using mixtures of recombinant Phox proteins and purified flavocytochrome b-245. Activation of oxidase activity required the simultaneous presence of p47(phox), flavocytochrome b-245, and the anionic amphiphile. The activation of electron transfer reactions was much more rapid when iodonitrotetrazolium violet was used as electron acceptor than when oxygen alone was the acceptor. We propose that this difference represents an intermediate activation state of NADPH oxidase in which electron flow can proceed from NADPH to enzyme flavin (and hence to iodonitrotetrazolium violet) but not from flavin to heme (or not between the hemes). A model for NADPH oxidase activation is presented that is consistent with these observations. (+info)
Carbocations in the synthesis of prostaglandins by the cyclooxygenase of PGH synthase? A radical departure!
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Evidence already available is used to demonstrate that although prostaglandin G/H synthase hydroxylates arachidonic acid through radical intermediates, it effects cyclizations through a carbocation center at C-10. This is produced following migration of H to the initial radical at C-13 and a 1epsilon oxidation. Under orbital symmetry control, the cyclizations can give only the ring size and trans stereochemistry actually observed. After cyclization, the H-shift reverses to take the sequence back into current radical theory for hydroxylation at C-15. Thus 10,10-difluoroarachidonic acid cannot be cyclized, although it can be hydroxylated. Acetylation of Ser516 in the isoform synthase-2 is considered to oppose carbocation formation and/or H-migration and so prevent cyclizations while permitting hydroxylations; the associated inversion of chirality at C-15 can then readily be accommodated without the change in conformation required by other schemes. Suicide inhibition occurs when carbocations form stable bonds upon (thermal) contact with adjacent heteroatoms, etc. Because the cyclooxygenase and peroxidase functions operate simultaneously through the same heme, phenol acts as reducing cosubstrate for the cyclooxygenase, thus enabling it to promote PGG2 production and protect the enzyme from oxidative destruction. (+info)
Identification of the proton pathway in bacterial reaction centers: inhibition of proton transfer by binding of Zn2+ or Cd2+.
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The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB(2) (QA-*QB-* + H+ --> QA(QBH)-, where QA is the primary quinone acceptor). Zn2+ and Cd2+ bound stoichiometrically to the RC (KD /= 10(2)-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D+*QAQB-* --> DQAQB suggests that the binding site is located far (>10 A) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB-* occurs. (+info)
Quantitative structure activity relationships for the electron transfer reactions of Anabaena PCC 7119 ferredoxin-NADP+ oxidoreductase with nitrobenzene and nitrobenzimidazolone derivatives: mechanistic implications.
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The steady state single electron reduction of polynitroaromatics by ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) from cyanobacterium Anabaena PCC 7119 has been studied and quantitative structure activity relationships are described. The solubility of the polynitroaromatics as well as their reactivity towards ferredoxin-NADP+ oxidoreductase are markedly higher than those for previously studied mononitroaromatics and this enabled the independent measurement of the kinetic parameters-k(cat) and Km. Interestingly, the natural logarithm of the bimolecular rate constant, k(cat)/Km, and also the natural logarithm of k(cat) correlate with the calculated energy of the lowest unoccupied molecular orbital of the polynitroaromatic substrates. The minimal kinetic model in line with these quantitative structure activity relationships is a ping-pong mechanism which includes substrate binding equilibria in the second half reaction. (+info)
Modulation of oxidative phosphorylation of human kidney 293 cells by transfection with the internal rotenone-insensitive NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae.
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In contrast to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which consists of at least 43 different subunits, the internal rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae is a single polypeptide enzyme. The NDI1 gene was stably transfected into the human embryonal kidney 293 (HEK 293) cells. The transfected NDI1 gene was then transcribed and translated in the HEK 293 cells to produce the functional enzyme. The immunochemical and immunofluorescence analyses indicated that the expressed Ndi1 polypeptide was located to the inner mitochondrial membranes. The expression of Ndi1 did not alter the content of existing complex I in the HEK 293 mitochondria, suggesting that the expressed Ndi1 enzyme does not displace the endogenous complex I. The NADH oxidase activity of the NDI1-transfected HEK 293 cells was not affected by rotenone but was inhibited by flavone. The ADP/O ratios coupled to NADH oxidation were lowered from 2.4 to 1.8 by NDI1-transfection while the ADP/O ratios coupled to succinate oxidation (1.6) were not changed. The NDI1-transfected HEK 293 cells were able to grow in media containing a complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. The potential usefulness of incorporating the Ndi1 protein into mitochondria of human cells is discussed. (+info)
The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.
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We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation. (+info)
PAS domains: internal sensors of oxygen, redox potential, and light.
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PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells. (+info)