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Dynamics of the K-B Proton Pathway in Cytochrome ba(3) from Thermus thermophilus
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Bern, Switzerland.
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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Number of Authors: 8
2017 (English)In: Israel Journal of Chemistry, ISSN 0021-2148, Vol. 57, no 5, 424-436 p.Article in journal (Refereed) Published
Abstract [en]

The ba(3) cytochrome c oxidase from Thermus thermophilus is a B-type oxygen-reducing heme-copper oxidase and a proton pump. It uses only one proton pathway for transfer of protons to the catalytic site, the K-B pathway. It was previously shown that the ba(3) oxidase has an overall similar reaction sequence to that in mitochondrial-like A-type oxidases. However, the timing of loading the pump site, and formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated variants in which two amino acids of the K-B proton pathway leading to the catalytic site were exchanged; Tyr-248 (located approximate to 23 angstrom below the active site towards the cytoplasm) in subunit I (Y248T) and Glu-15 (approximate to 26 angstrom below the active site, approximate to 16 angstrom from Tyr-248) in subunit II (E15(II)Q). Even though the overall catalytic turnover in these two variants is similar and very low (<1% of wildtype), the substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site. The results indicate that the Glu-15(II) is the only essentially crucial residue of the K-B pathway, but that the Tyr-248 also plays a distinct role in defining an internal proton donor and controlling the dynamics of proton transfer to the pump site and the catalytic site.

Place, publisher, year, edition, pages
2017. Vol. 57, no 5, 424-436 p.
Keyword [en]
heme-copper oxidases, cytochrome c oxidase, proton transfer, electron transfer, membrane protein, respiration, redox reaction, metalloprotein, cytochrome aa(3), cytochrome cbb(3)
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-144708DOI: 10.1002/ijch.201600136ISI: 000401329000009OAI: oai:DiVA.org:su-144708DiVA: diva2:1128034
Available from: 2017-07-21 Created: 2017-07-21 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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