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Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Number of Authors: 3
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 3, e0152745Article in journal (Refereed) Published
Abstract [en]

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 N2O via the following reaction: 2NO+2e(-)+2H(+) -> N2O+H2O. 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.

Place, publisher, year, edition, pages
2016. Vol. 11, no 3, e0152745
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Biological Sciences
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URN: urn:nbn:se:su:diva-130138DOI: 10.1371/journal.pone.0152745ISI: 000373121800132PubMedID: 27030968OAI: oai:DiVA.org:su-130138DiVA: diva2:929051
Available from: 2016-05-17 Created: 2016-05-09 Last updated: 2016-05-17Bibliographically approved

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ter Beek, JosyÄdelroth, Pia
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