Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: ter Beek, Josy, Krause, Nils, Ädelroth, Pia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
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
_version_ 1782424743705575424
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
work_keys_str_mv AT terbeekjosy investigatingtheprotondonorinthenoreductasefromparacoccusdenitrificans
AT krausenils investigatingtheprotondonorinthenoreductasefromparacoccusdenitrificans
AT adelrothpia investigatingtheprotondonorinthenoreductasefromparacoccusdenitrificans