Department of Biochemistry

Biochemistry of denitrification bacteria

H3>Research team
  • Igor Kučera,
  • Pavel Bouchal
  • Vojtěch Sedláček

Project summary

Denitrification is an anaerobic respiratory process carried out by microorganisms able to use N-oxides as alternative electron acceptors to oxygen. Through denitrifcation, N-oxoanions nitrate and nitrite are converted to dinitrogen and, to a lesser extent, nitrous oxide, which is a potent greenhouse gas and protective ozone layer destroyer. We work with Paracoccus denitrificans as a model denitrifying organism. This bacterium normally inhabits ecosystems like soil, water and sewage sludge. Aerobically growing cells have a respiratory chain closely resembling that of mitochondria with respect to the presence of the respiratory complexes I – IV and a ubiquinone homologue with ten isoprene units in the side chain (UQ-10). Being unable to ferment, the bacterium´s strategy to survive anaerobiosis is to gain energy from denitrification. For this purpose it produces four enzymes (nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase) that catalyze stepwise conversion of the ubiquitously present nitrate to the gaseous dinitrogen.

Our research group seeks to understand molecular mechanisms of some interesting oxidoreduction reactions, their metabolic roles of and their affection following exposure of cells to various external factors. We use a wide variety of approaches ranging from molecular biology tools (gene cloning and sequencing, cassette and site-directed mutagenesis, promoter-reporter gene fusions, quantitative RT-PCR), biochemical methods (protein purification, activity assays) and structure determination via X-ray crystallography to whole cell studies with the wild-type and mutant strains.

Most important results or innovations

  • Distribution of electron flux in the branched respiratory chain. The electron transport system in the cytoplasmic membrane of P. denitrificans terminates in three distinct oxidases accomplishing oxygen reduction to water and in four denitrification enzymes catalyzing the reaction sequence nitrate -> nitrite -> nitric oxide -> nitrous oxide -> dinitrogen. We would like to shed more light on a quite complicated problem of mutual interactions among these enzymes, resulting from their competition for common electron donors and from the inhibitory effects of nitric oxide. One way is the use of specific mutant strains.
  • Significance of the rate of nitrate transport into cells. Nitrate must cross the cell membrane prior to interaction with the first enzyme of the denitrification pathway. This transfer is mediated by a specific nitrate transporter and is often considered a bottleneck in the whole denitrification process. We are currently addressing this possibility by using strains expressing exclusively either membrane-bound cytoplasmically oriented (Nar) or periplasmic (Nap) nitrate reductase or by means of nitrate transporter inhibitors.
  • Flavin-dependent oxidoreductases FerA and FerB. P. denitrificans possesses two soluble enzymes, FerA and FerB, capable of reducing a number of ferric complexes at the expense of NADH. Both proteins interact with flavins, but in a different manner. FerA obligatorily uses flavin as a co-substrate and hence can be classified as an NADH:flavin oxidoreductase. On the contrary, FerB contains a non-covalently bound redox active flavin coenzyme which transfers electrons from NAD(P)H to various acceptors like quinones, Fe(III) complexes or chromate. Recently we have succeeded in cloning and overexpressing the ferB gene, crystallization of a selenomethionyl derivative of the protein and determination of its crystal structure. The major benefit from the obtained structural data will be to enable subsequent studies on structure-function relationships. A special effort will also be made to clarify the role of FerA and FerB proteins in cellular metabolism.
  • Response of bacterial proteome to growth conditions changes. We are currently using 2D gel electrophoresis and densitometry to compare protein levels in various strains growing under aerobic, semianaerobic and anaerobic conditions. As a part of this work, we will also look at the effects of compounds that induce oxidative stress with the aim to detect and identify the involved proteins and genes.

Selected publications

Financial support

Mechanisms involved in regulation of respiratory systems in denitrification bacteria by evironmental factors. GACR 203/01/1589

Structure, function and regulation of FerB, a broad-specificity bacterial oxidoreductase of a potential ecotechnological relevance. GACR 525/07/1069

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