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Nitric Oxide Reductase from Paracoccus denitrificans: A Proton Transfer Pathway from the “Wrong” Side
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Responsible organisation
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Denitrification is an anaerobic process performed by several soil bacteria as an alternative to aerobic respiration. A key-step in denitrification (the N-N-bond is made) is catalyzed by nitric oxide reductase (NOR); 2NO + 2e- + 2H+ → N2O + H2O. NOR from Paracoccus denitrificans is a member of the heme copper oxidase superfamily (HCuOs), where the mitochondrial cytochrome c oxidase is the classical example. NOR is situated in the cytoplasmic membrane and can, as a side reaction, catalyze the reduction of oxygen to water.

NORs have properties that make them divergent members of the HCuOs; the reactions they catalyze are not electrogenic and they do not pump protons. They also have five strictly conserved glutamates in their catalytic subunit (NorB) that are not conserved in the ‘classical’ HCuOs. It has been asked whether the protons used in the reaction really come from the periplasm and if so how do the protons proceed through the protein into the catalytic site?

In order to find out whether the protons are taken from the periplasm or the cytoplasm and in order to pinpoint the proton-route in NorB, we studied electron- and proton transfer during a single- as well as multiple turnovers, using time resolved optical spectroscopy. Wild type NOR and several variants of the five conserved glutamates were investigated in their solubilised form or/and reconstituted into vesicles.

The results demonstrate that protons needed for the reaction indeed are taken from the periplasm and that all but one of the conserved glutamates are crucial for the oxidative phase of the reaction that is limited by proton uptake to the active site.

In this thesis it is proposed, using a model of NorB, that two of the glutamates are located at the entrance of the proton pathway which also contains two of the other glutamates close to the active site.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik , 2008. , 113 p.
Keyword [en]
denitrification, nitric oxide reductase, heme copper oxidase superfamily, divergent member, proton transfer, electron transfer, single turnover, spectroscopy, periplasm, glutamate, proton pathway
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-8171ISBN: 978-91-7155-740-7 (print)OAI: oai:DiVA.org:su-8171DiVA: diva2:199755
Public defence
2008-10-17, Magnélisalen, Kemiska övnigslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-09-25 Created: 2008-09-25 Last updated: 2015-09-24Bibliographically approved
List of papers
1. Electron/Proton Coupling in Bacterial Nitric Oxide Reductase during Reduction of Oxygen
Open this publication in new window or tab >>Electron/Proton Coupling in Bacterial Nitric Oxide Reductase during Reduction of Oxygen
2005 In: Biochemistry, Vol. 44, no 31, 10711-10719 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25461 (URN)
Note
Part of urn:nbn:se:su:diva-8171Available from: 2008-09-25 Created: 2008-09-25Bibliographically approved
2. A pathway for protons in nitric oxide reductase from Paracoccus denitrificans
Open this publication in new window or tab >>A pathway for protons in nitric oxide reductase from Paracoccus denitrificans
Show others...
2007 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1767, no 5, 362-373 p.Article in journal (Refereed) Published
Abstract [en]

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N2O (2NO + 2e(-) + 2H(+) -> N2O + H2O) as part of the denitriffication process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O-2-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O-2, we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O-2 resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O-2 shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O-2 as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O-2 show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.

Keyword
proton transfer, electron transfer, proteoliposomes, flow-flash, non-heme iron, nitric oxide, oxygen, homology modeling, sequence alignments
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-25432 (URN)10.1016/j.bbabio.2007.03.006 (DOI)000246654200003 ()
Available from: 2008-09-04 Created: 2008-09-04 Last updated: 2011-10-06Bibliographically approved
3. Defining the Proton Entry Point in the Bacterial Respiratory Nitric-oxide Reductase
Open this publication in new window or tab >>Defining the Proton Entry Point in the Bacterial Respiratory Nitric-oxide Reductase
Show others...
2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 283, no 7, 3839-3845 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25463 (URN)10.1074/jbc.M704615200 (DOI)000253083500018 ()
Note
Part of urn:nbn:se:su:diva-8171Available from: 2008-09-25 Created: 2008-09-25 Last updated: 2009-05-12Bibliographically approved
4. Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase
Open this publication in new window or tab >>Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase
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2009 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 103, no 5, 845-850 p.Article in journal (Refereed) Published
Abstract [en]

The c-type nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans is an integral membrane protein that catalyzes NO reduction; 2NO+2e(-)+2H(+)-->N(2)O+H(2)O. It is also capable of catalyzing the reduction of oxygen to water, albeit more slowly than NO reduction. cNORs are divergent members of the heme-copper oxidase superfamily (HCuOs) which reduce NO, do not pump protons, and the reaction they catalyse is non-electrogenic. All known cNORs have been shown to have five conserved glutamates (E) in the catalytic subunit, by P. denitrificans numbering, the E122, E125, E198, E202 and E267. The E122 and E125 are presumed to face the periplasm and the E198, E202 and E267 are located in the interior of the membrane, close to the catalytic site. We recently showed that the E122 and E125 define the entry point of the proton pathway leading from the periplasm into the active site [U. Flock, F.H. Thorndycroft, A.D. Matorin, D.J. Richardson, N.J. Watmough, P. Adelroth, J. Biol. Chem. 283 (2008) 3839-3845]. Here we present results from the reaction between fully reduced NOR and oxygen on the alanine variants of the E198, E202 and E267. The initial binding of O(2) to the active site was unaffected by these mutations. In contrast, proton uptake to the bound O(2) was significantly inhibited in both the E198A and E267A variants, whilst the E202A NOR behaved essentially as wildtype. We propose that the E198 and E267 are involved in terminating the proton pathway in the region close to the active site in NOR.

Keyword
Proton transfer, Electron transfer, Ligand binding, Flow-flash
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-34709 (URN)10.1016/j.jinorgbio.2009.02.008 (DOI)000265758200024 ()19332356 (PubMedID)
Available from: 2010-01-11 Created: 2010-01-11 Last updated: 2015-10-19Bibliographically approved

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