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The timing of heme incorporation into yeast cytochrome b
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

During oxidative phosphorylation electrons are transferred from NADH and succinate to the final electron acceptor oxygen by the complexes of the respiratory chain. These complexes carry redox active prosthetic groups that allow the transfer of electrons. Cytochrome b of the bc1 complex is encoded in the mitochondrial genome and acquires two heme b cofactors during its biogenesis. In this work we aimed to understand the mechanism and timing of cytochrome b hemylation. We provide evidence that cytochrome b present in the first bc1 complex assembly intermediate that contains the assembly factors Cbp3-Cbp6 and Cbp4 carries heme. This demonstrates that heme acquisition occurs very early during cytochrome b biogenesis. Moreover, by analyzing cytochrome b mutants lacking either of the two heme moieties, we reveal an obligate order of heme insertion into cytochrome b and suggest an incorporation mode from the intermembrane space. We propose a model in which Cbp3-Cbp6 keeps cytochrome b in a conformation allowing heme acquisition. Upon heme insertion, cytochrome b most likely undergoes a conformational change that enables binding of Cbp4, a pre-requisite for further assembly. Cbp4 thus might exhibit a proofreading function in hemylation.

National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-81053OAI: oai:DiVA.org:su-81053DiVA: diva2:559211
Available from: 2012-10-08 Created: 2012-10-08 Last updated: 2012-10-09Bibliographically approved
In thesis
1. Early steps in the biogenesis of the bc1 complex in yeast mitochondria: The role of the Cbp3-Cbp6 complex in cytochrome b synthesis and assembly
Open this publication in new window or tab >>Early steps in the biogenesis of the bc1 complex in yeast mitochondria: The role of the Cbp3-Cbp6 complex in cytochrome b synthesis and assembly
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The inner membrane of mitochondria harbors the complexes of the respiratory chain and the ATP synthase, which perform the key metabolic process oxidative phosphorylation. These complexes are composed of subunits from two different genetic origins: the majority of constituents is synthesized on cytosolic ribosomes and imported into mitochondria, but a handful of proteins, which represent core catalytic subunits, are encoded in the organellar DNA and translated on mitochondrial ribosomes. Using yeast as a model organism, I investigated the mitochondrial ribosomal tunnel exit, the region of the ribosome where the nascent chain emerges and that in cytosolic ribosomes serves as a platform to bind biogenesis factors that help the newly synthesized protein to mature. This study provided insights into the structural composition of this important site of mitochondrial ribosomes and revealed the positioning of Cbp3 at the tunnel exit region, a chaperone required specifically for the assembly of the bc1 complex. In my further work I found that Cbp3 structurally and functionally forms a tight complex with Cbp6 and that this complex exhibits fundamental roles in the biogenesis of cytochrome b, the mitochondrially encoded subunit of the bc1 complex. Bound to the ribosome, Cbp3-Cbp6 stimulates translation of the cytochrome b mRNA (COB mRNA). Cbp3-Cbp6 then binds the fully synthesized cytochrome b, thereby stabilizing and guiding it further through bc1 complex assembly. The next steps involve the recruitment of the assembly factor Cbp4 to the Cbp3-Cbp6/cytochrome b complex and presumably acquisition of two redox active heme b cofactors. During further assembly Cbp3-Cbp6 is released from cytochrome b, can again bind to the ribosome and activate further rounds of COB mRNA translation. The dual role of Cbp3-Cbp6 in both translation and assembly allows the complex to act as a regulatory switch to modulate the level of cytochrome b synthesis in response to the bc1 complex assembly process.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2012. 54 p.
Keyword
mitochondria, respiratory chain assembly, bc1 complex, mitochondrial gene expression, translational regulation, hemylation
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-81033 (URN)978-91-7447-534-0 (ISBN)
Public defence
2012-12-07, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2012-11-15 Created: 2012-10-08 Last updated: 2012-11-07Bibliographically approved

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