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Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase
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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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.

Place, publisher, year, edition, pages
2009. Vol. 103, no 5, 845-850 p.
Keyword [en]
Proton transfer, Electron transfer, Ligand binding, Flow-flash
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-34709DOI: 10.1016/j.jinorgbio.2009.02.008ISI: 000265758200024PubMedID: 19332356OAI: oai:DiVA.org:su-34709DiVA: diva2:285333
Available from: 2010-01-11 Created: 2010-01-11 Last updated: 2017-12-12Bibliographically approved
In thesis
1. 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
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. Kinetics of proton and electron transfer in heme-copper oxidases
Open this publication in new window or tab >>Kinetics of proton and electron transfer in heme-copper oxidases
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Heme-copper oxidases are transmembrane proteins that are found in aerobic and anaerobic respiratory chains. During aerobic respiration, these enzymes reduce dioxygen to water. The energy released in the reaction is used to transport protons across a biological membrane. Stored as proton electrochemical gradient, the energy can be used to regenerate ATP. It is known that aa3 oxidases, which are the most common oxidases, transport pumped protons and protons used for the catalytic reaction using two proton pathways. However, the molecular mechanism of pumping is still being debated.

When oxygen is available in very small quantities, oxygen reductases with high affinity for oxygen are expressed by organisms like Thermus thermophilus. The proton pumping mechanism in the ba3 oxidase is slightly different from that of aa3 oxidases as this enzyme only uses a single proton uptake pathway. Here we analyzed the reaction mechanism of ba3 oxidase and found evidence that the first proton taken up by the four-electron reduced ba3 oxidase is transferred to a site distant from the catalytic site, the pump site, and that only every second proton taken up from solution is pumped. Data obtained from studies using site-directed mutagenesis and flow-flash spectroscopy suggest a probable location of the pump site.

Under anaerobic conditions, some organisms are able to generate a proton- motive force using nitrate and nitrite as electron acceptors. In this process, the cytotoxic reaction intermediate nitric oxide is produced. Nitric oxide reductase (NOR), a deviant heme-copper oxidase that reduces NO to the rather harmless N2O, does not pump any protons. The catalytic mechanism of nitric oxide reduction by NOR is very poorly understood.

Here we demonstrate that substrate inhibition, which occurs in NOR from Paracoccus denitrificans above 5 μM NO, can already be observed before the electrons from the low-spin hemes re-distribute to the active site. Furthermore, we found that a single specific proton pathway is used for proton-transfer leading from the periplasm to the active site. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015. 64 p.
Keyword
Heme-copper oxidase, electron transfer, proton transfer, nitric oxide reductase, ba3 oxidase, flow-flash, laser-flash photolysis
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-119996 (URN)978-91-7649-263-5 (ISBN)
Public defence
2015-11-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2015-10-30 Created: 2015-08-31 Last updated: 2015-10-22Bibliographically approved

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