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.
- 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
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.
- 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
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.
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 genetika chemie metabolismus MeSH
- cytochromy c genetika chemie metabolismus MeSH
- financování organizované MeSH
- inhibitory enzymů metabolismus MeSH
- oxidace-redukce MeSH
- Paracoccus denitrificans chemie metabolismus MeSH
- respirační komplex III genetika chemie metabolismus MeSH
- statická elektřina MeSH
