Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.
- Klíčová slova
- Paracoccus denitrificans, antioxidant enzyme, chromate reductase, flavoprotein, superoxide reductase,
- MeSH
- arginin genetika MeSH
- flavinmononukleotid chemie genetika MeSH
- flaviny genetika metabolismus MeSH
- FMN-reduktasa chemie genetika MeSH
- katalytická doména genetika MeSH
- kinetika MeSH
- konformace proteinů * MeSH
- krystalografie rentgenová MeSH
- oxidace-redukce MeSH
- oxidoreduktasy chemie genetika MeSH
- Paracoccus denitrificans chemie enzymologie MeSH
- sekvence aminokyselin genetika MeSH
- superoxidy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- arginin MeSH
- chromate reductase MeSH Prohlížeč
- ferric citrate iron reductase MeSH Prohlížeč
- flavinmononukleotid MeSH
- flaviny MeSH
- FMN-reduktasa MeSH
- oxidoreduktasy MeSH
- superoxidy MeSH
A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.
- Klíčová slova
- Metabolomics, Nucleotides, Paracoccus denitrificans, Stem cells, System biology,
- MeSH
- acetylkoenzym A analýza MeSH
- adenosintrifosfát analýza MeSH
- chemické techniky analytické metody normy MeSH
- cytidintrifosfát analýza MeSH
- embryonální kmenové buňky chemie MeSH
- guanosintrifosfát analýza MeSH
- lidé MeSH
- limita detekce MeSH
- Paracoccus denitrificans chemie MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acetylkoenzym A MeSH
- adenosintrifosfát MeSH
- cytidintrifosfát MeSH
- guanosintrifosfát MeSH
The main aim of this work was to demonstrate the applicability of capillary zone electrophoresis in combination with field enhanced sample stacking in targeted metabolome analyses of adenine nucleotides--AMP, ADP, ATP, coenzymes NAD(+), NADP(+) and their reduced forms in Paracoccus denitrificans. Sodium carbonate/hydrogencarbonate buffer (100 mM, pH 9.6) with the addition of beta-CD at a concentration of 10 mM was found to be an effective BGE for their separation within 20 min. Besides this, special attention was paid to the development of the procedure for the extraction of specific metabolites from the bacterium P. denitrificans. This procedure was not only optimised to achieve the highest metabolite yields but also to obtain a sample that was fully compatible with the online preconcetration strategy used. The developed methodology was finally applied in a study of the bacterium P. denitrificans at various stages of the active respiratory chain.
- MeSH
- adeninnukleotidy analýza metabolismus MeSH
- časové faktory MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- koenzymy analýza metabolismus MeSH
- metabolom * MeSH
- Paracoccus denitrificans chemie enzymologie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adeninnukleotidy MeSH
- koenzymy MeSH
The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.
- MeSH
- antibakteriální látky metabolismus MeSH
- antimycin A metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- cytochromy c chemie genetika metabolismus MeSH
- inhibitory enzymů metabolismus MeSH
- oxidace-redukce MeSH
- Paracoccus denitrificans chemie metabolismus MeSH
- respirační komplex III chemie genetika metabolismus MeSH
- statická elektřina MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- antimycin A MeSH
- bakteriální proteiny MeSH
- cytochromy c MeSH
- inhibitory enzymů MeSH
- respirační komplex III MeSH
Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients was carried out on total cell lysates and membrane fractions of Paracoccus denitrificans with the aim to characterize differences in protein expression during growth under aerobic and various anaerobic conditions (with nitrate, nitrite or nitrous oxide). Comparative image analysis of the protein pattern revealed several subgroups of the total 800 protein spots resolved that were characteristically induced or repressed in response to individual electron acceptors. The respiratory inhibitor azide also exerted a profound influence upon cellular protein composition. However, since most of the proteins showing an altered expression pattern in cells growing on oxygen differed from those in cells growing on nitrite, we suppose that azide acts mainly indirectly, possibly by influencing other cellular signals. Limited information on the P. denitrificans genome has precluded the identification of more than eight protein spots as yet. A public accessible P. denitrificans 2-DE protein database is currently built up at http://www.mpiib-berlin.mpg.de/2D-PAGE.
- MeSH
- 2D gelová elektroforéza MeSH
- azid sodný chemie MeSH
- bakteriální proteiny chemie metabolismus MeSH
- databáze proteinů MeSH
- elektrony * MeSH
- hmotnostní spektrometrie MeSH
- inhibitory enzymů chemie MeSH
- membránové proteiny chemie MeSH
- nitritreduktasy chemie metabolismus MeSH
- Paracoccus denitrificans chemie cytologie fyziologie MeSH
- proteom analýza MeSH
- subcelulární frakce chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- azid sodný MeSH
- bakteriální proteiny MeSH
- inhibitory enzymů MeSH
- membránové proteiny MeSH
- nitritreduktasy MeSH
- proteom MeSH
The levels of nitrate in denitrifying cells of Paracoccus denitrificans were determined by centrifugation through silicone oil into phosphoric acid and ion-exchange HPLC analysis of the cell lysates, using [14C]sucrose to correct for the trapped external medium. Introduction of oxygen brought about a significant upward shift in the intracellular nitrate concentration. This result calls into question the current thinking that oxygen blocks nitrate respiration primarily due to the inhibition of nitrate transport into the cell.
- MeSH
- biologický transport fyziologie MeSH
- dusičnany analýza metabolismus MeSH
- hodnotící studie jako téma MeSH
- kyslík farmakologie MeSH
- Paracoccus denitrificans chemie účinky léků metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- dusičnany MeSH
- kyslík MeSH
Unlike the uncoupler carbonyl cyanide 3-chlorophenyl-hydrazone, the respiratory inhibitors CN-, N3-, NO2- and rotenone enhanced the formation of nitrate and nitrite reductases in highly aerated cultures of the Paracoccus denitrificans ex-conjugant PD1222 (pRW2A/FF). A maximal effect was observed at concentrations partly blocking electron transport to O2. The level of beta-galactosidase reporting the activity of an Fnr-like regulatory protein showed a similar concentration dependency. It is concluded that oxygen is sensed by Fnr in an indirect way, possibly via the redox state of a cellular component.
- MeSH
- aerobióza MeSH
- anaerobióza MeSH
- azidy farmakologie MeSH
- bakteriální proteiny metabolismus MeSH
- beta-galaktosidasa biosyntéza metabolismus MeSH
- dusík metabolismus MeSH
- kyanidy farmakologie MeSH
- nitrátreduktasy biosyntéza metabolismus MeSH
- nitritreduktasy biosyntéza metabolismus MeSH
- oxid dusičitý farmakologie MeSH
- oxidace-redukce MeSH
- oxygenasy antagonisté a inhibitory metabolismus MeSH
- Paracoccus denitrificans chemie účinky léků fyziologie MeSH
- proteiny obsahující železo a síru * MeSH
- proteiny z Escherichia coli * MeSH
- rotenon farmakologie MeSH
- spotřeba kyslíku účinky léků MeSH
- transport elektronů účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- azidy MeSH
- bakteriální proteiny MeSH
- beta-galaktosidasa MeSH
- dusík MeSH
- FNR protein, E coli MeSH Prohlížeč
- kyanidy MeSH
- nitrátreduktasy MeSH
- nitritreduktasy MeSH
- oxid dusičitý MeSH
- oxygenasy MeSH
- proteiny obsahující železo a síru * MeSH
- proteiny z Escherichia coli * MeSH
- rotenon MeSH
