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Functional Role of Thr-312 and Thr-315 in the Proton-Transfer Pathway in ba3 Cytochrome c Oxidase from Thermus thermophilus
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
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2010 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, nr 33, s. 7033-7039Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Cytochrome ba3 from Thermus thermophilus is a member of the family of B-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like A-type enzymes. Recently, it was suggested that the ba3 oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the K-pathway in the A-type oxidases [Chang, H.-Y., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 16169−16173]. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the time-resolved reaction between fully reduced cytochrome ba3 and O2 in variants where Thr-312 and Thr-315 were modified. While in the A-type oxidases this reaction is essentially unchanged in variants with the K-pathway modified, in the Thr-312 → Ser variant in the ba3 oxidase both reactions associated with proton uptake from solution, the PR → F and F → O transitions, were slowed compared to those of wild-type ba3. The observed time constants were slowed 3-fold (for PR → F, from 60 to 170 μs in the wild type) and 30-fold (for F → O, from 1.1 to 40 ms). In the Thr-315 → Val variant, the F → O transition was 5-fold slower (5 ms) than for the wild-type oxidase, whereas the PR → F transition displayed an essentially unchanged time constant. However, the uptake of protons from solution was a factor of 2 slower and decoupled from the optical PR → F transition. Our results thus show that proton uptake is significantly and specifically inhibited in the two variants, strongly supporting the suggested involvement of T312 and T315 in the transfer of protons to the active site during O2 reduction in the ba3 oxidase.

Ort, förlag, år, upplaga, sidor
2010. Vol. 49, nr 33, s. 7033-7039
Nationell ämneskategori
Biologiska vetenskaper
Forskningsämne
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-50523DOI: 10.1021/bi100749pISI: 000280855200007OAI: oai:DiVA.org:su-50523DiVA, id: diva2:381636
Anmärkning
authorCount :8Tillgänglig från: 2010-12-28 Skapad: 2010-12-28 Senast uppdaterad: 2022-02-24Bibliografiskt granskad
Ingår i avhandling
1. Proton transfer in nitric oxide reducing heme-copper oxidases
Öppna denna publikation i ny flik eller fönster >>Proton transfer in nitric oxide reducing heme-copper oxidases
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. s. 76
Nyckelord
heme-copper oxidases, nitric oxide, proton transfer, electron transfer, proton-transfer pathway
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-62893 (URN)978-91-7447-377-3 (ISBN)
Disputation
2011-11-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2011-10-20 Skapad: 2011-10-03 Senast uppdaterad: 2022-02-24Bibliografiskt granskad

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Reimann, Joachimvon Ballmoos, ChristophBrzezinski, PeterÄdelroth, Pia

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