scholarly journals Mitochondria recycle nitrite back to the bioregulator nitric monoxide.

2000 ◽  
Vol 47 (4) ◽  
pp. 913-921 ◽  
Author(s):  
H Nohl ◽  
K Staniek ◽  
B Sobhian ◽  
S Bahrami ◽  
H Redl ◽  
...  
Keyword(s):  
Respiratory Chain ◽  
Paracoccus Denitrificans ◽  
Hypovolemic Shock ◽  
Heme Iron ◽  
Nitrite Reduction ◽  
No Donors ◽  
Mammalian Mitochondria ◽  
Membrane Bound ◽  
Chemical Sequestration ◽  
Nitric Monoxide

Nitric monoxide (NO) exerts a great variety of physiological functions. L-Arginine supplies amino groups which are transformed to NO in various NO-synthase-active isoenzyme complexes. NO-synthesis is stimulated under various conditions increasing the tissue of stable NO-metabolites. The major oxidation product found is nitrite. Elevated nitrite levels were reported to exist in a variety of diseases including HIV, reperfusion injury and hypovolemic shock. Denitrifying bacteria such as Paracoccus denitrificans have a membrane bound set of cytochromes (cyt cd1, cyt bc) which were shown to be involved in nitrite reduction activities. Mammalian mitochondria have similar cytochromes which form part of the respiratory chain. Like in bacteria quinols are used as reductants of these types of cytochromes. The observation of one-e- divergence from this redox-couple to external dioxygen made us to study whether this site of the respiratory chain may also recycle nitrite back to its bioactive form NO. Thus, the aim of the present study was therefore to confirm the existence of a reductive pathway which reestablishes the existence of the bioregulator NO from its main metabolite NO2-. Our results show that respiring mitochondria readily reduce added nitrite to NO which was made visible by nitrosylation of deoxyhemoglobin. The adduct gives characteristic triplet-ESR-signals. Using inhibitors of the respiratory chain for chemical sequestration of respiratory segments we were able to identify the site where nitrite is reduced. The results confirm the ubiquinone/cyt be1 couple as the reductant site where nitrite is recycled. The high affinity of NO to the heme-iron of cytochrome oxidase will result in an impairment of mitochondrial energy-production. "Nitrite tolerance" of angina pectoris patients using NO-donors may be explained in that way.

scholarly journals Modifications of the Aerobic Respiratory Chain of Paracoccus Denitrificans in Response to Superoxide Oxidative Stress

2019 ◽  
Vol 7 (12) ◽  
pp. 640 ◽  
Author(s):  
Vojtěch Sedláček ◽  
Igor Kučera
Keyword(s):  
Oxidative Stress ◽  
Respiratory Chain ◽  
Nadh Dehydrogenase ◽  
Paracoccus Denitrificans ◽  
Mrna Level ◽  
Oxygen Species ◽  
Stress Perception ◽  
Iron Sulfur Clusters ◽  
Iron Sulfur ◽  
Mammalian Mitochondria

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.

scholarly journals A new assay for nitric oxide reductase reveals two conserved glutamate residues form the entrance to a proton-conducting channel in the bacterial enzyme

2006 ◽  
Vol 401 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Faye H. Thorndycroft ◽  
Gareth Butland ◽  
David J. Richardson ◽  
Nicholas J. Watmough
Keyword(s):  
Nitric Oxide ◽  
Paracoccus Denitrificans ◽  
Nitric Oxide Reductase ◽  
Transmembrane Helices ◽  
Level Of Activity ◽  
Bacterial Enzyme ◽  
Cytoplasmic Membranes ◽  
Membrane Bound ◽  
Proton Movement ◽  
High Level

A specific amperometric assay was developed for the membrane-bound NOR [NO (nitric oxide) reductase] from the model denitrifying bacterium Paracoccus denitrificans using its natural electron donor, pseudoazurin, as a co-substrate. The method allows the rapid and specific assay of NO reduction catalysed by recombinant NOR expressed in the cytoplasmic membranes of Escherichia coli. The effect on enzyme activity of substituting alanine, aspartate or glutamine for two highly conserved glutamate residues, which lie in a periplasmic facing loop between transmembrane helices III and IV in the catalytic subunit of NOR, was determined using this method. Three of the substitutions (E122A, E125A and E125D) lead to an almost complete loss of NOR activity. Some activity is retained when either Glu122 or Glu125 is substituted with a glutamine residue, but only replacement of Glu122 with an aspartate residue retains a high level of activity. These results are interpreted in terms of these residues forming the mouth of a channel that conducts substrate protons to the active site of NOR during turnover. This channel is also likely to be that responsible in the coupling of proton movement to electron transfer during the oxidation of fully reduced NOR with oxygen [U. Flock, N. J. Watmough and P. Ädelroth (2005) Biochemistry 44, 10711–10719].

scholarly journals A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c550 and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd1 Nitrite Reductase

2003 ◽  
Vol 185 (21) ◽  
pp. 6308-6315 ◽  
Author(s):  
Isobel V. Pearson ◽  
M. Dudley Page ◽  
Rob J. M. van Spanning ◽  
Stuart J. Ferguson
Keyword(s):  
Nitrite Reductase ◽  
Paracoccus Denitrificans ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Electron Mediator ◽  
Intact Cells ◽  
Cytochrome Cd1 ◽  
Membrane Bound ◽  
Electron Carriers

ABSTRACT In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc 1 complex to the periplasmic nitrite reductase, cytochrome cd 1. The periplasmic protein cytochrome c 550 has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome c 550 and pseudoazurin are alternative electron carriers from the cytochrome bc 1 complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c 550. The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c 550 or both these proteins. We concluded that pseudoazurin and cytochrome c 550 are the alternative electron mediator proteins between the cytochrome bc 1 complex and the cytochrome cd 1-type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

The ‘strict’ anaerobeDesulfovibrio gigascontains a membrane-bound oxygen-reducing respiratory chain

FEBS Letters ◽  
2001 ◽  
Vol 496 (1) ◽  
pp. 40-43 ◽  
Author(s):  
Rita S Lemos ◽  
Cláudio M Gomes ◽  
Margarida Santana ◽  
Jean LeGall ◽  
António V Xavier ◽  
...  
Keyword(s):  
Respiratory Chain ◽  
Membrane Bound

A composite biochemical system for bacterial nitrate and nitrite assimilation as exemplified by Paracoccus denitrificans

2011 ◽  
Vol 435 (3) ◽  
pp. 743-753 ◽  
Author(s):  
Andrew J. Gates ◽  
Victor M. Luque-Almagro ◽  
Alan D. Goddard ◽  
Stuart J. Ferguson ◽  
M. Dolores Roldán ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Nitrogen Source ◽  
Nitrite Reductase ◽  
Paracoccus Denitrificans ◽  
Gene Clusters ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Nitrite Reduction ◽  
Anaerobic Growth ◽  
Nitrate And Nitrite

The denitrifying bacterium Paracoccus denitrificans can grow aerobically or anaerobically using nitrate or nitrite as the sole nitrogen source. The biochemical pathway responsible is expressed from a gene cluster comprising a nitrate/nitrite transporter (NasA), nitrite transporter (NasH), nitrite reductase (NasB), ferredoxin (NasG) and nitrate reductase (NasC). NasB and NasG are essential for growth with nitrate or nitrite as the nitrogen source. NADH serves as the electron donor for nitrate and nitrite reduction, but only NasB has a NADH-oxidizing domain. Nitrate and nitrite reductase activities show the same Km for NADH and can be separated by anion-exchange chromatography, but only fractions containing NasB retain the ability to oxidize NADH. This implies that NasG mediates electron flux from the NADH-oxidizing site in NasB to the sites of nitrate and nitrite reduction in NasC and NasB respectively. Delivery of extracellular nitrate to NasBGC is mediated by NasA, but both NasA and NasH contribute to nitrite uptake. The roles of NasA and NasC can be substituted during anaerobic growth by the biochemically distinct membrane-bound respiratory nitrate reductase (Nar), demonstrating functional overlap. nasG is highly conserved in nitrate/nitrite assimilation gene clusters, which is consistent with a key role for the NasG ferredoxin, as part of a phylogenetically widespread composite nitrate and nitrite reductase system.

Sign in / Sign up

Export Citation Format

Share Document