Paracoccus denitrificans is a strictly respiring bacterium with a core respiratory chain similar to that of mammalian mitochondria. As such, it continuously produces and has to cope with superoxide and other reactive oxygen species. In this work, the effects of artificially imposed superoxide stress on electron transport were examined. Exposure of aerobically growing cells to paraquat resulted in decreased activities of NADH dehydrogenase, succinate dehydrogenase, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) oxidase. Concomitantly, the total NAD(H) pool size in cells was approximately halved, but the NADH/NAD+ ratio increased twofold, thus partly compensating for inactivation losses of the dehydrogenase. The inactivation of respiratory dehydrogenases, but not of TMPD oxidase, also took place upon treatment of the membrane fraction with xanthine/xanthine oxidase. The decrease in dehydrogenase activities could be fully rescued by anaerobic incubation of membranes in a mixture containing 2-mercaptoethanol, sulfide and ferrous iron, which suggests iron-sulfur clusters as targets for superoxide. By using cyanide titration, a stress-sensitive contribution to the total TMPD oxidase activity was identified and attributed to the cbb3-type terminal oxidase. This response (measured by both enzymatic activity and mRNA level) was abolished in a mutant defective for the FnrP transcription factor. Therefore, our results provide evidence of oxidative stress perception by FnrP.

• Keywords
• FnrP transcription factor, NADH dehydrogenase, iron–sulfur cluster, succinate dehydrogenase, superoxide, terminal oxidase,
• Publication type
• Journal Article MeSH

The review briefly summarizes current knowledge of the bacterial nitric-oxide reductase (NOR). This membrane enzyme consists of two subunits, the smaller one contains haem C and the larger one two haems B and nonhaem iron. The protein sequence and structure of metal centres demonstrate the relationship of NOR to the family of terminal oxidases. The binuclear Fe-Fe reaction centre, consisting of antiferromagnetically coupled haem B and nonhaem iron, is analogous to Fe-Cu centre of terminal oxidases. The data on the structure and function of NOR and terminal oxidases suggest that all these enzymes are closely evolutionally related. The catalytic properties are determined most of all by the relatively high toxicity of nitric oxide as a substrate and the resulting strong need to maintain its concentration at nanomolar levels. A kinetic model of the action of the enzyme comprises substrate inhibition. NOR does not conserve the free energy of nitric oxide reduction because it does not work as a proton pump and, moreover, the protons coming into the reaction are taken from periplasm, i.e. they do not cross the membrane.

• MeSH
• Bacteria enzymology   MeSH
• Nitric Oxide metabolism   MeSH
• Oxidoreductases chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• nitric-oxide reductase MeSH Browser
• Nitric Oxide MeSH
• Oxidoreductases MeSH

The metabolism of nitric oxide in Paracoccus denitrificans has been studied using a Clark-type electrode. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and the SH reagent N-ethylmaleimide, both of which released nitric oxide from cells respiring nitrite, were found to be efficient inhibitors of nitric oxide reductase activity. Control experiments with another uncoupler, pentachlorophenol, showed that the inhibitory effect of CCCP was not the result of a decrease in membrane potential. The denitrification pathway in cells with partly inhibited nitric oxide reductase, or in a reconstituted system containing purified nitric reductase and membrane vesicles, exhibited marked sustained oscillations of nitric oxide concentration. The occurrence of the oscillations was strictly dependent on the initial concentration of nitrite. The observed oscillatory kinetics is considered to reflect two regulatory signals destabilizing the denitrification pathway, namely the inhibition of nitric oxide reductase by nitric oxide and/or by nitrite.

• MeSH
• Biological Transport MeSH
• Cell Membrane drug effects   physiology   MeSH
• Cytochrome c Group metabolism   MeSH
• Ethylmaleimide pharmacology   MeSH
• Carbonyl Cyanide m-Chlorophenyl Hydrazone pharmacology   MeSH
• Kinetics MeSH
• Membrane Potentials drug effects   MeSH
• Nitrite Reductases isolation & purification   metabolism   MeSH
• Nitric Oxide metabolism   MeSH
• Oxidoreductases isolation & purification   metabolism   MeSH
• Paracoccus denitrificans metabolism   physiology   MeSH
• Pentachlorophenol pharmacology   MeSH
• Uncoupling Agents pharmacology   MeSH
• Oxygen Consumption drug effects   MeSH
• Electron Transport drug effects   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytochrome c Group MeSH
• Ethylmaleimide MeSH
• Carbonyl Cyanide m-Chlorophenyl Hydrazone MeSH
• nitric-oxide reductase MeSH Browser
• Nitrite Reductases MeSH
• Nitric Oxide MeSH
• Oxidoreductases MeSH
• Pentachlorophenol MeSH
• Uncoupling Agents MeSH