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The Nitric-oxide Reductase from Paracoccus denitrificans Uses a Single Specific Proton Pathway
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.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 42, 30626-30635 p.Article in journal (Refereed) Published
Abstract [en]

The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

Place, publisher, year, edition, pages
2013. Vol. 288, no 42, 30626-30635 p.
Keyword [en]
Bioenergetics, Electron Transfer Complex, Electron Transfer, Enzyme Kinetics, Membrane Biophysics, Nitric Oxide, Proton Transport, Flow-Flash, Heme-Copper Oxidase, Kinetic Isotope Effect
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-98606DOI: 10.1074/jbc.M113.497347ISI: 000329868100063PubMedID: 24014024OAI: oai:DiVA.org:su-98606DiVA: diva2:684579
Available from: 2014-01-08 Created: 2014-01-08 Last updated: 2017-12-06Bibliographically approved
In thesis
1. 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)
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Available from: 2015-10-30 Created: 2015-08-31 Last updated: 2015-10-22Bibliographically approved

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