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Artificial Electron Acceptor (artificial + electron_acceptor)
Selected AbstractsTwo W-containing formate dehydrogenases (CO2 -reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidansFEBS JOURNAL, Issue 11 2003Frank A. M. De Bok Two formate dehydrogenases (CO2 -reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2 -reduction rates, and low Km values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U·mg,1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U·mg,1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor. The specific CO2 -reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U·mg,1 and 89 U·mg,1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 ± 3, 56 ± 2 and 19 ± 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 ± 3 kDa and a 33 ± 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH-1 contains at least four [2Fe-2S] clusters per molecule and additionally paramagnetically coupled [4Fe-4S] clusters. FDH-2 contains at least two [4Fe-4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters. [source] Rates of electron transport in the thylakoid membranes of isolated, illuminated chloroplasts are enhanced in the presence of ammonium chlorideBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 6 2003Rob L. Dean Abstract The rate of flow of electrons from water to an artificial electron acceptor, dichlorophenolindophenol (DCPIP), through photosystem II in the thylakoid membranes of isolated chloroplasts is greatly enhanced in the presence of 10 mM ammonium chloride. Rate enhancement depends on irradiance levels. Uncoupling reagents like ammonium chloride prevent the formation of a proton gradient across the thylakoid membrane and consequently remove a constraint on the rate of electron transport. The mode of action of ammonium chloride is explained. Evidence obtained using an oxygen electrode that DCPIP itself also partially uncouples the system is presented as background information for instructors. Suggestions on how this reaction may be used in laboratory classes for students from high school to the senior undergraduate level are included. [source] Hydrogen production by photoautotrophic sulfur-deprived Chlamydomonas reinhardtii pre-grown and incubated under high lightBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Irina V. Tolstygina Abstract We have previously demonstrated that Chlamydomonas reinhardtii can produce hydrogen under strictly photoautotrophic conditions during sulfur deprivation [Tsygankov et al. (2006); Int J Hydrogen Energy 3:1574,1584]. The maximum hydrogen photoproduction was achieved by photoautotrophic cultures pre-grown under a low light regime (25 µE,m,2,s,1). We failed to establish sustained hydrogen production from cultures pre-grown under high light (100 µE,m,2,s,1). A new approach for sustained hydrogen production by these cultures is presented here. Assuming that stable and reproducible transition to anerobiosis as well as high starch accumulation are important for hydrogen production, the influence of light intensity and dissolved oxygen concentration during the oxygen evolving stage of sulfur deprivation were investigated in cultures pre-grown under high light. Results showed that light higher than 175 µE,m,2,s,1 during sulfur deprivation induced reproducible transition to anerobiosis, although the total amount of starch accumulation and hydrogen production were insignificant. The potential PSII activity measured in the presence of an artificial electron acceptor (DCBQ) and an inhibitor of electron transport (DBMIB) did not change in cultures pre-grown under 20 µE,m,2,s,1 and incubated under 150 µE,m,2,s,1 during sulfur deprivation. In contrast, the potential PSII activity decreased in cultures pre-grown under 100 µE,m,2,s,1 and incubated under 420 µE,m,2,s,1. This indicates that cultures grown under higher light experience irreversible inhibition of PSII in addition to reversible down regulation. High dissolved O2 content during the oxygen evolving stage of sulfur deprivation has a negative regulatory role on PSII activity. To increase hydrogen production by C. reinhardtii pre-grown under 100 µE,m,2,s,1, cultures were incubated under elevated PFD and decreased oxygen pressure during the oxygen evolving stage. These cultures reproducibly reached anaerobic stage, accumulated significant quantities of starch and produced significant quantities of H2. It was found that elevation of pH from 7.4 to 7.7 during the oxygen producing stage of sulfur deprivation led to a significant increase of accumulated starch. Thus, control of pH during sulfur deprivation is a possible way to further optimize hydrogen production by photoautotrophic cultures. Biotechnol. Bioeng. 2009;102: 1055,1061. © 2008 Wiley Periodicals, Inc. [source] Inhibition of pea ferredoxin,NADP(H) reductase by Zn-ferrocyanideFEBS JOURNAL, Issue 22 2004Daniela L. Catalano Dupuy Ferredoxin,NADP(H) reductases (FNRs) represent a prototype of enzymes involved in numerous metabolic pathways. We found that pea FNR ferricyanide diaphorase activity was inhibited by Zn2+ (Ki 1.57 µm). Dichlorophenolindophenol diaphorase activity was also inhibited by Zn2+ (Ki 1.80 µm), but the addition of ferrocyanide was required, indicating that the inhibitor is an arrangement of both ions. Escherichia coli FNR was also inhibited by Zn-ferrocyanide, suggesting that inhibition is a consequence of common structural features of these flavoenzymes. The inhibitor behaves in a noncompetitive manner for NADPH and for artificial electron acceptors. Analysis of the oxidation state of the flavin during catalysis in the presence of the inhibitor suggests that the electron-transfer process between NADPH and the flavin is not significantly altered, and that the transfer between the flavin and the second substrate is mainly affected. Zn-ferrocyanide interacts with the reductase, probably increasing the accessibility of the prosthetic group to the solvent. Ferredoxin reduction was also inhibited by Zn-ferrocyanide in a noncompetitive manner, but the observed Ki was about nine times higher than those for the diaphorase reactions. The electron transfer to Anabaena flavodoxin was not affected by Zn-ferrocyanide. Binding of the apoflavodoxin to the reductase was sufficient to overcome the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners may induce a conformational change in the reductase that alters or completely prevents the inhibitory effect. [source] Exploring the primary electron acceptor (QA)-site of the bacterial reaction center from Rhodobacter sphaeroidesFEBS JOURNAL, Issue 4 2002Binding mode of vitamin K derivatives The functional replacement of the primary ubiquinone (QA) in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides with synthetic vitamin K derivatives has provided a powerful tool to investigate the electron transfer mechanism. To investigate the binding mode of these quinones to the QA binding site we have determined the binding free energy and charge recombination rate from QA, to D+ (kAD) of 29 different 1,4-naphthoquinone derivatives with systematically altered structures. The most striking result was that none of the eight tested compounds carrying methyl groups in both positions 5 and 8 of the aromatic ring exhibited functional binding. To understand the binding properties of these quinones on a molecular level, the structures of the reaction center-naphthoquinone complexes were predicted with ligand docking calculations. All protein,ligand structures show hydrogen bonds between the carbonyl oxygens of the quinone and AlaM260 and HisM219 as found for the native ubiquinone-10 in the X-ray structure. The center-to-center distance between the naphthoquinones at QA and the native ubiquinone-10 at QB (the secondary electron acceptor) is essentially the same, compared to the native structure. A detailed analysis of the docking calculations reveals that 5,8-disubstitution prohibits binding due to steric clashes of the 5-methyl group with the backbone atoms of AlaM260 and AlaM249. The experimentally determined binding free energies were reproduced with an rmsd of ,,4 kJ·mol,1 in most cases providing a valuable tool for the design of new artificial electron acceptors and inhibitors. [source] Pyrroloquinoline quinone-dependent carbohydrate dehydrogenase: Activity enhancement and the role of artificial electron acceptorsBIOTECHNOLOGY JOURNAL, Issue 8 2010Juozas Kulys Professor Abstract Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH) offers a variety of opportunities for applications, e.g. in highly sensitive biosensors and electrosynthetic reactions. Here we report on the acceleration (up to 4.9 x 104 -fold) of enzymatic ferricyanide reduction by artificial redox mediators (enhancers). The reaction mechanism includes reduction of the PQQ-GDH by glucose followed by oxidation of the reduced PQQ cofactor with either ferricyanide or a redox mediator. A synergistic effect occurs through the oxidation of a reduced mediator by ferricyanide. Using kinetic description of the coupled reaction, the second order rate constant for the reaction of an oxidized mediator with the reduced enzyme cofactor (kox) can be calculated. For different mediators this value is 2.2 x 106,1.6 x 108 M -1s -1 at pH 7.2 and 25°C. However, no correlation of the rate constant with the midpoint redox potential of the mediator could be established. For low-potential mediators the synergistic effect is proportional to the ratio of kox(med)/kox(ferricyanide), whereas for the high-potential mediators the effect depends on both this ratio and the concentration of the oxidized mediator, which can be calculated from the Nernst equation. The described effect can be applied in various ways, e.g. for substrate reactivity determination, electrosynthetic PQQ cofactor regeneration or building of new highly sensitive biosensors. [source] | ||||||||||