Functional role of Thr-312 and Thr-315 in the proton-transfer pathway in ba 3 cytochrome c oxidase from thermus thermophilus

Irina Smirnova, Joachim Reimann, Christoph Von Ballmoos, Hsin Yang Chang, Robert B. Gennis, James A. Fee, Peter Brzezinski, Pia Ädelroth

Research output: Contribution to journalArticlepeer-review

Abstract

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 P R → 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.

Original languageEnglish (US)
Pages (from-to)7033-7039
Number of pages7
JournalBiochemistry
Volume49
Issue number33
DOIs
StatePublished - Aug 24 2010

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Functional role of Thr-312 and Thr-315 in the proton-transfer pathway in ba <sub>3</sub> cytochrome c oxidase from thermus thermophilus'. Together they form a unique fingerprint.

Cite this