Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characteristics

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].

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Functional interactions between nitrite reductase and nitric oxide reductase from Paracoccus denitrificans

Scientific Reports ◽

10.1038/s41598-019-53553-z ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Ingrid Albertsson ◽

Johannes Sjöholm ◽

Josy ter Beek ◽

Nicholas J. Watmough ◽

Jerker Widengren ◽

...

Keyword(s):

Nitric Oxide ◽

Nitrite Reductase ◽

Paracoccus Denitrificans ◽

Affinity Constant ◽

Nitric Oxide Reductase ◽

Terminal Electron Acceptor ◽

Model Soil ◽

Transient Interactions ◽

Electron Donation

AbstractDenitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd1 nitrite reductase (cd1NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd1NiR, presumably because cd1NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd1NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd1NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd1NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.

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Kinetic Analysis of Substrate Inhibition in Nitric Oxide Reductase of Paracoccus denitrificans

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1999.1245 ◽

1999 ◽

Vol 262 (2) ◽

pp. 562-564 ◽

Cited By ~ 14

Author(s):

Marek Koutný ◽

Igor Kučera

Keyword(s):

Nitric Oxide ◽

Kinetic Analysis ◽

Paracoccus Denitrificans ◽

Substrate Inhibition ◽

Nitric Oxide Reductase

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Proton transfer in bacterial nitric oxide reductase

Biochemical Society Transactions ◽

10.1042/bst0340188 ◽

2006 ◽

Vol 34 (1) ◽

pp. 188-190 ◽

Cited By ~ 13

Author(s):

U. Flock ◽

J. Reimann ◽

P. Ädelroth

Keyword(s):

Nitric Oxide ◽

Proton Transfer ◽

Structural Model ◽

No Reduction ◽

Paracoccus Denitrificans ◽

Proton Donor ◽

Nitric Oxide Reductase ◽

Reduction Of No ◽

Proton Coupled Electron Transfer ◽

Electron Reduction

The NOR (nitric oxide reductase) from Paracoccus denitrificans catalyses the two-electron reduction of NO to N2O (2NO+2H++2e−→N2O+H2O). The NOR is a divergent member of the superfamily of haem-copper oxidases, oxygen-reducing enzymes which couple the reduction of oxygen with translocation of protons across the membrane. In contrast, reduction of NO catalysed by NOR is non-electrogenic which, since electrons are supplied from the periplasmic side of the membrane, implies that the protons needed for NO reduction are also taken from the periplasm. Thus NOR must contain a proton-transfer pathway leading from the periplasmic side of the membrane into the catalytic site. The proton pathway has not been identified, and the mechanism and timing of proton transfer during NO reduction is unknown. To address these questions, we have studied the reaction between NOR and the chemically less reactive oxidant O2 [Flock, Watmough and Ädelroth (2005) Biochemistry 44, 10711–10719]. When fully reduced NOR reacts with O2, proton-coupled electron transfer occurs in a reaction that is rate-limited by internal proton transfer from a group with a pKa of 6.6. This group is presumably an amino acid residue close to the active site that acts as a proton donor also during NO reduction. The results are discussed in the framework of a structural model that identifies possible candidates for the proton donor as well as for the proton-transfer pathway.

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