ABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Iron-Sulfur Proteins , Oxygen Consumption/drug effects , Paracoccus denitrificans/chemistry , Paracoccus denitrificans/drug effects , Aerobiosis , Anaerobiosis , Azides/pharmacology , Cyanides/pharmacology , Electron Transport/drug effects , Nitrate Reductases/biosynthesis , Nitrate Reductases/metabolism , Nitrite Reductases/biosynthesis , Nitrite Reductases/metabolism , Nitrogen/metabolism , Nitrogen Dioxide/pharmacology , Oxidation-Reduction , Oxygenases/antagonists & inhibitors , Oxygenases/metabolism , Paracoccus denitrificans/physiology , Rotenone/pharmacology , beta-Galactosidase/biosynthesis , beta-Galactosidase/metabolismABSTRACT
The role of periplasmic cytochrome c in the denitrification pathway has been investigated using a wild-type and/or a cytochrome c deficient strain of Paracoccus denitrificans. The reconstitution experiments with the isolated proteins showed that bacterial cytochrome c-550 restored the electron transport from the cytoplasmic membrane to soluble nitrite reductase (cytochrome cd1). In response to decreased aeration lasting 3 h, the HUUG25 strain synthesized nitrous-oxide reductase significantly starved of electrons from the respiratory chain and only very small amounts of soluble cytochrome c. The membrane-bound part of the respiratory chain catalyzing the reduction of soluble cytochrome c resembled an autologous region in wild-type cells kinetically and by its sensitivity to antimycin. In the periplasmic fraction obtained from anaerobically grown wild-type cells N2O caused the reoxidation of endogenous cytochrome(s) c previously reduced by N,N,N',N' tetramethyl-p-phenylenediamine plus ascorbate. All these results indicate the involvement of soluble cytochrome(s) c as the electron donor(s) for the reduction of NO2- and N2O in the periplasmic space of cells.
Subject(s)
Cytochrome c Group/physiology , Nitrites/metabolism , Nitrous Oxide/metabolism , Paracoccus denitrificans/metabolism , Electron Transport , Genes, Bacterial , Kinetics , Mutation , Oxidation-Reduction , Oxygen/metabolism , Paracoccus denitrificans/enzymology , Paracoccus denitrificans/genetics , Spectrum AnalysisABSTRACT
A new method of determination of nitrate was developed, utilizing the nitrate reductase activity of Paracoccus denitrificans in which a further reduction of nitrate is blocked either by a mutation affecting formation of cytochromes c or by inhibition of the electron flow to nitrite reductase by mucidin. After deproteinization of the sample with zinc acetate the nitrite produced is determined colorimetrically.
