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A pathway for protons in nitric oxide 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.
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2007 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, 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.

Place, publisher, year, edition, pages
2007. Vol. 1767, no 5, 362-373 p.
Keyword [en]
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: urn:nbn:se:su:diva-25432DOI: 10.1016/j.bbabio.2007.03.006ISI: 000246654200003OAI: oai:DiVA.org:su-25432DiVA: diva2:199697
Available from: 2008-09-04 Created: 2008-09-04 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Experimental studies of proton translocation reactions in biological systems: Electrogenic events in heme-copper oxidases
Open this publication in new window or tab >>Experimental studies of proton translocation reactions in biological systems: Electrogenic events in heme-copper oxidases
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O2 to H2O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the aa3-type oxidases, to which cytochrome c oxidase (CytcO) belongs, which uses energy released in the O2-reduction for proton pumping. The bacterial nitric oxide reductases (NORs) have been identified as divergent members of the HCuO-superfamily and are involved in the denitrification pathway where they catalyze the reduction of NO to NO2. Although as exergonic as O2-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the cbb3-type oxidases are the closest relatives to the NORs and as such provide a link between the aa3 oxidases and the NORs. The cbb3 oxidases have been shown to pump protons with nearly the same efficiency as the aa3 oxidases, despite low sequence similarity.

This thesis is focused on measurements of membrane potential generating reactions during catalysis in the CytcO and the cbb3 oxidase from Rhodobacter sphaeroides, and the NOR from Paracoccus denitrificans, using a time resolved electrometric technique. The pH dependence of the membrane potential generation in CytcO showed that only one proton is taken up and that no protons are pumped, at high pH. An additional kinetic phase was also detected at high pH that presumably originates to from charge-transfer within the K-pathway. Possible reasons for uncoupling, and the extent of charge-transfer, were studied using structural variants of CytcO. The measurements established that electrons and protons are taken up from the same side of the membrane in NOR. In addition, the directionality for proton uptake in cbb3 oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O2-reduction.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 62 p.
Keyword
heme-copper oxidase, cytochrome c oxidase, nitric oxide reductase, cbb3-type oxidase, proton pumping, uncoupling, charge-transfer, electrogenic event, flow-flash
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-8147 (URN)978-91-7155-712-4 (ISBN)
Public defence
2008-09-12, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00
Opponent
Supervisors
Available from: 2008-09-04 Created: 2008-09-04Bibliographically approved
2. Nitric Oxide Reductase from Paracoccus denitrificans: A Proton Transfer Pathway from the “Wrong” Side
Open this publication in new window or tab >>Nitric Oxide Reductase from Paracoccus denitrificans: A Proton Transfer Pathway from the “Wrong” Side
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
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:nbn:se:su:diva-8171 (URN)978-91-7155-740-7 (ISBN)
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
3. Proton transfer in nitric oxide reducing heme-copper oxidases
Open this publication in new window or tab >>Proton transfer in nitric oxide reducing heme-copper oxidases
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Heme-copper oxidases (HCuOs) are best known as terminal oxidases in the aerobic respiratory chain, in which they catalyze the reduction of oxygen to water. By receiving protons and electrons from opposite sides of the membrane as well as pumping protons, HCuOs contribute to the electrochemical proton gradient over the membrane that can be used for ATP synthesis. Divergent members of the HCuO superfamily are nitric oxide reductases (NORs) that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O) as part of the denitrification process, an alternative respiratory pathway.

The first part of the thesis focuses on electron and proton transfer reactions that are associated with the reductive conversion of NO to N2O and O2 to H2O by the NOR from Paracoccus denitrificans. Our data show that proton uptake in NOR is not electrogenic (protons and electrons are taken up from the same side of the membrane) and that no protons are pumped. Also, structural variants have been investigated and the results suggest a role for these residues in proton transfer. Further, we show that lowering the pH leads to a higher NO reduction rate, while this effect is partially counteracted by a larger degree of substrate inhibition at low pH.

The second part deals with proton transfer and electrical potential generation in the reaction between the cbb3 oxidase from Rhodobacter sphaeroides and O2 or NO. Our data show that NO reduction by cbb3 oxidase is not coupled to proton translocation and that the direction of proton uptake is dependent on substrate. Our findings suggest that the proton pumping mechanism in HCuOs is incompatible with NO reduction intermediates.

Finally, experiments on structural variants of the ba3 oxidase from Thermus thermophilus indicate a functional role for the inspected residues in proton transfer and support the suggestion that a single proton-transfer pathway is used in the ba3 oxidase.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 76 p.
Keyword
heme-copper oxidases, nitric oxide, proton transfer, electron transfer, proton-transfer pathway
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62893 (URN)978-91-7447-377-3 (ISBN)
Public defence
2011-11-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2011-10-20 Created: 2011-10-03 Last updated: 2011-10-17Bibliographically approved

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