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Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans
Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript (c), which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71(c)D; exchange of Glu-71 in NorC for an Asp). Bacterial NO reductases (NORs) are integral membrane...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816578/ https://www.ncbi.nlm.nih.gov/pubmed/27030968 http://dx.doi.org/10.1371/journal.pone.0152745 |
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author | ter Beek, Josy Krause, Nils Ädelroth, Pia |
author_facet | ter Beek, Josy Krause, Nils Ädelroth, Pia |
author_sort | ter Beek, Josy |
collection | PubMed |
description | Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript (c), which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71(c)D; exchange of Glu-71 in NorC for an Asp). Bacterial NO reductases (NORs) are integral membrane proteins from the heme-copper oxidase superfamily. Most heme-copper oxidases are proton-pumping enzymes that reduce O(2) as the last step in the respiratory chain. With electrons from cytochrome c, NO reductase (cNOR) from Paracoccus (P.) denitrificans reduces NO to N(2)O via the following reaction: 2NO+2e(-)+2H(+)→N(2)O+H(2)O. Although this reaction is as exergonic as O(2)-reduction, cNOR does not contribute to the electrochemical gradient over the membrane. This means that cNOR does not pump protons and that the protons needed for the reaction are taken from the periplasmic side of the membrane (since the electrons are donated from this side). We previously showed that the P. denitrificans cNOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance. Here we further strengthened the evidence in support of this pathway. Our further aim was to define the continuation of the pathway and the immediate proton donor for the active site. To this end, we investigated the region around the calcium-binding site and both propionates of heme b(3) by site directed mutagenesis. Changing single amino acids in these areas often had severe effects on cNOR function, with many variants having a perturbed active site, making detailed analysis of proton transfer properties difficult. Our data does however indicate that the calcium ligation sphere and the region around the heme b(3) propionates are important for proton transfer and presumably contain the proton donor. The possible evolutionary link between the area for the immediate donor in cNOR and the proton loading site (PLS) for pumped protons in oxygen-reducing heme-copper oxidases is discussed. |
format | Online Article Text |
id | pubmed-4816578 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-48165782016-04-14 Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans ter Beek, Josy Krause, Nils Ädelroth, Pia PLoS One Research Article Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript (c), which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71(c)D; exchange of Glu-71 in NorC for an Asp). Bacterial NO reductases (NORs) are integral membrane proteins from the heme-copper oxidase superfamily. Most heme-copper oxidases are proton-pumping enzymes that reduce O(2) as the last step in the respiratory chain. With electrons from cytochrome c, NO reductase (cNOR) from Paracoccus (P.) denitrificans reduces NO to N(2)O via the following reaction: 2NO+2e(-)+2H(+)→N(2)O+H(2)O. Although this reaction is as exergonic as O(2)-reduction, cNOR does not contribute to the electrochemical gradient over the membrane. This means that cNOR does not pump protons and that the protons needed for the reaction are taken from the periplasmic side of the membrane (since the electrons are donated from this side). We previously showed that the P. denitrificans cNOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance. Here we further strengthened the evidence in support of this pathway. Our further aim was to define the continuation of the pathway and the immediate proton donor for the active site. To this end, we investigated the region around the calcium-binding site and both propionates of heme b(3) by site directed mutagenesis. Changing single amino acids in these areas often had severe effects on cNOR function, with many variants having a perturbed active site, making detailed analysis of proton transfer properties difficult. Our data does however indicate that the calcium ligation sphere and the region around the heme b(3) propionates are important for proton transfer and presumably contain the proton donor. The possible evolutionary link between the area for the immediate donor in cNOR and the proton loading site (PLS) for pumped protons in oxygen-reducing heme-copper oxidases is discussed. Public Library of Science 2016-03-31 /pmc/articles/PMC4816578/ /pubmed/27030968 http://dx.doi.org/10.1371/journal.pone.0152745 Text en © 2016 ter Beek et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article ter Beek, Josy Krause, Nils Ädelroth, Pia Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans |
title | Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans |
title_full | Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans |
title_fullStr | Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans |
title_full_unstemmed | Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans |
title_short | Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans |
title_sort | investigating the proton donor in the no reductase from paracoccus denitrificans |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816578/ https://www.ncbi.nlm.nih.gov/pubmed/27030968 http://dx.doi.org/10.1371/journal.pone.0152745 |
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