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Proton transfer in the quinol dependent nitric oxide reductase from geobacillus stearothermophilus during reduction of oxygen
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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2012 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1817, no 10, 1914-1920 p.Article in journal (Refereed) Published
Abstract [en]

Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246]. In this study, we have investigated the reaction between this gNOR and oxygen. Our results show that, like some cNORs, the C. stearothermophilus gNOR is capable of 02 reduction with a turnover of similar to 3 electrons s(-1) at 40 degrees C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) gNOR reacts with oxygen in a reaction with a time constant of 1.8 ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between gNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in gNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Place, publisher, year, edition, pages
2012. Vol. 1817, no 10, 1914-1920 p.
Keyword [en]
Heme-copper oxidase, Proton transfer pathway, Non-heme iron, Flow-flash, Carbon monoxide
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-81832DOI: 10.1016/j.bbabio.2012.04.007ISI: 000307918200028OAI: oai:DiVA.org:su-81832DiVA: diva2:565372
Note

AuthorCount:7;

Available from: 2012-11-07 Created: 2012-11-01 Last updated: 2017-09-20Bibliographically approved
In thesis
1. Proton pathways in energy conversion: K-pathway analogs in O2- and NO-reductases
Open this publication in new window or tab >>Proton pathways in energy conversion: K-pathway analogs in O2- and NO-reductases
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oxygen and nitric oxide reductases are enzymes found in aerobic and anaerobic respiration, respectively. Both enzyme groups belong to the superfamily of Heme-Copper Oxidases, which is further divided into several subgroups: oxygen-reducing enzymes into A-, B- and C-type and nitric oxide reductases into qNORs and cNORs. Oxygen reducing enzymes use the energy released from oxygen reduction to take up electrons and protons from different sides of the membrane. Additionally, protons are pumped. These processes produce a membrane potential, which is used by the ATP-synthase to produce ATP, the universal energy currency of the cell. Nitric oxide reductases are not known to conserve the energy from nitric oxide reduction, although the reaction is highly exergonic.

Here, the detailed mechanism of a B-type oxidase is studied with special interest in an element involved in proton pumping (proton loading site, PLS). The study supports the hypothesis that the PLS is protonated in one and deprotonated in the consecutive step of the oxidative catalytic cycle, and that a proton is pumped during the final oxidation phase. It further strengthens the previous suggestion that the PLS is a cluster instead of a single residue or heme propionate. Additionally, it is proposed that the residue Asp372, which is in vicinity of the heme a3 propionates previously suggested as PLS, is part of this cluster. In another study, we show that the Glu15II at the entry of the proton pathway in the B-type oxidase is the only crucial residue for proton uptake, while Tyr248 is or is close to the internal proton donor responsible for coupling proton pumping to oxygen reduction.

The thesis also includes studies on the mechanism and electrogenicity of qNOR. We show that there is a difference in the proton-uptake reaction between qNOR and the non-electrogenic homolog cNOR, hinting at a different reaction mechanism. Further, studies on a qNOR from a different host showed that qNOR is indeed electrogenic. This surprising result opens up new discussions on the evolution of oxygen and nitric oxide reductases, and about how energy conservation can be achieved.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017. 66 p.
Keyword
heme-copper oxidase, cytochrome c oxidase, membrane protein, respiration, electron transfer, proton transfer, redox reaction, metalloprotein, non-heme iron, cytochrome ba3, flow-flash, carbon monoxide, liposome, respiratory control ratio
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147267 (URN)978-91-7649-986-3 (ISBN)978-91-7649-987-0 (ISBN)
Public defence
2017-11-09, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
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Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2017-10-17 Created: 2017-09-20 Last updated: 2017-10-04Bibliographically approved

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