Dataset Information

Functional role of Thr-312 and Thr-315 in the proton-transfer pathway in ba3 Cytochrome c oxidase from Thermus thermophilus.

ABSTRACT: Cytochrome ba(3) 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 ba(3) 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 ba(3) and O(2) 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 ba(3) 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 ba(3). The observed time constants were slowed approximately 3-fold (for P(R) --> F, from 60 to approximately 170 mus in the wild type) and approximately 30-fold (for F --> O, from 1.1 to approximately 40 ms). In the Thr-315 --> Val variant, the F --> O transition was approximately 5-fold slower (5 ms) than for the wild-type oxidase, whereas the P(R) --> 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 P(R) --> 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 O(2) reduction in the ba(3) oxidase.

SUBMITTER: Smirnova I

PROVIDER: S-EPMC2932646 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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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.

Smirnova Irina I Reimann Joachim J von Ballmoos Christoph C Chang Hsin-Yang HY Gennis Robert B RB Fee James A JA Brzezinski Peter P Adelroth Pia P

Biochemistry 20100801 33

Cytochrome ba(3) 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 ba(3) 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 pat ...[more]

PMID: 20677778

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Project description:The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO) superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H(+) and e(-) transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba(3)-type cytochrome c oxidase from Thermus thermophilus at 1.8 Å resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O(2)-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the Cu(B) atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fe(a3) and Cu(B) atoms that is best modeled as peroxide. The structure of ba(3)-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba(3)-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.

Dataset Information

Functional role of Thr-312 and Thr-315 in the proton-transfer pathway in ba3 Cytochrome c oxidase from Thermus thermophilus.

Publications

Functional role of Thr-312 and Thr-315 in the proton-transfer pathway in ba3 Cytochrome c oxidase from Thermus thermophilus.

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