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      Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc 1 function in vivo

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          Highlights

          • We used hybrid fusion bc 1 complex to test inter-monomer electron transfer in vivo.

          • Cross-inactivated complexes were able to sustain photoheterotrophic growth.

          • Inter-monomer electron transfer supports catalytic cycle in vivo.

          • bc 1 dimer is functional even when cytochrome b subunits come from different species.

          Abstract

          Electronic connection between Q o and Q i quinone catalytic sites of dimeric cytochrome bc 1 is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the latter remains unclear. Here, using a series of mutated hybrid cytochrome bc 1-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary.

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          Most cited references26

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          Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria.

          On the basis of x-ray diffraction data to a resolution of 2.9 angstroms, atomic models of most protein components of the bovine cytochrome bc1 complex were built, including core 1, core 2, cytochrome b, subunit 6, subunit 7, a carboxyl-terminal fragment of cytochrome c1, and an amino-terminal fragment of the iron-sulfur protein. The positions of the four iron centers within the bc1 complex and the binding sites of the two specific respiratory inhibitors antimycin A and myxothiazol were identified. The membrane-spanning region of each bc1 complex monomer consists of 13 transmembrane helices, eight of which belong to cytochrome b. Closely interacting monomers are arranged as symmetric dimers and form cavities through which the inhibitor binding pockets can be accessed. The proteins core 1 and core 2 are structurally similar to each other and consist of two domains of roughly equal size and identical folding topology.
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            Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment.

            The cytochrome bc(1) complex is part of the energy conversion machinery of the respiratory and photosynthetic electron transfer chains. This integral membrane protein complex catalyzes electron transfer from ubiquinol to cytochrome c. It couples the electron transfer to the electrogenic translocation of protons across the membrane via a so-called Q cycle mechanism. The cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae was crystallized together with a bound antibody Fv fragment. The structure was determined at 2.3 A resolution using multiple isomorphous replacement, and refined to a crystallographic R factor of 22.2% (R(free) = 25.4%). The complex is present as a homodimer. Each 'monomer' of the refined model includes 2178 amino acid residues of subunits COR1, QCR2, COB, CYT1, RIP1, QCR6, QCR7, QCR8 and QCR9 of the cytochrome bc(1) complex and of the polypeptides V(H) and V(L) of the Fv fragment, the cofactors heme b(H), heme b(L), heme c(1), the [2Fe-2S] cluster and 346 water molecules. The Fv fragment binds to the extrinsic domain of the [2Fe-2S] Rieske protein and is essential for formation of the crystal lattice. The approach to crystallize membrane proteins as complexes with specific antibody fragments appears to be of general importance. The structure of the yeast cytochrome bc(1) complex reveals in detail the binding sites of the natural substrate coenzyme Q6 and the inhibitor stigmatellin. Buried water molecules close to the binding sites suggest possible pathways for proton uptake and release. A comparison with other cytochrome bc(1) complexes shows features that are specific to yeast.
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              Structure and function of cytochrome bc complexes.

              The cytochrome bc complexes represent a phylogenetically diverse group of complexes of electron-transferring membrane proteins, most familiarly represented by the mitochondrial and bacterial bc1 complexes and the chloroplast and cyanobacterial b6f complex. All these complexes couple electron transfer to proton translocation across a closed lipid bilayer membrane, conserving the free energy released by the oxidation-reduction process in the form of an electrochemical proton gradient across the membrane. Recent exciting developments include the application of site-directed mutagenesis to define the role of conserved residues, and the emergence over the past five years of X-ray structures for several mitochondrial complexes, and for two important domains of the b6f complex.
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                Author and article information

                Contributors
                Journal
                Biochem Biophys Res Commun
                Biochem. Biophys. Res. Commun
                Biochemical and Biophysical Research Communications
                Academic Press
                0006-291X
                1090-2104
                22 August 2014
                22 August 2014
                : 451
                : 2
                : 270-275
                Affiliations
                Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland
                Author notes
                [* ]Corresponding author. artur.osyczka@ 123456uj.edu.pl
                Article
                S0006-291X(14)01366-7
                10.1016/j.bbrc.2014.07.117
                4152375
                25089001
                fabc12dd-7f95-4377-bc67-281bed363775
                © 2014 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

                History
                : 16 July 2014
                Categories
                Article

                Biochemistry
                cytochrome bc1,mitochondrial complex iii,hybrid fusion protein,electron transfer,energy conversion

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