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Mutation of a single residue in the ba(3) oxidase specifically impairs protonation of the pump site
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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2015 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 11, 3397-3402 p.Article in journal (Refereed) Published
Abstract [en]

The ba(3)-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O-2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba(3) oxidase where a putative pump site was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O-2 reduction. The results from our studies show that proton uptake to the pump site (time constant similar to 65 mu s in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of similar to 1.2 ms was slowed to similar to 8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba(3) cytochrome c oxidase.

Place, publisher, year, edition, pages
2015. Vol. 112, no 11, 3397-3402 p.
Keyword [en]
cytochrome c oxidase, membrane protein, respiration, cytochrome aa(3), electron transfer
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-116611DOI: 10.1073/pnas.1422434112ISI: 000351060000072PubMedID: 25733886OAI: oai:DiVA.org:su-116611DiVA: diva2:809119
Note

AuthorCount:6;

Available from: 2015-04-30 Created: 2015-04-22 Last updated: 2017-12-04Bibliographically 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)
Opponent
Supervisors
Available from: 2015-10-30 Created: 2015-08-31 Last updated: 2015-10-22Bibliographically approved
2. 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)
Opponent
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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