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. 2018 Feb 6;26(2):312-319.e3.
doi: 10.1016/j.str.2017.12.014. Epub 2018 Jan 26.

Structure of the Deactive State of Mammalian Respiratory Complex I

James N Blaza  1 , Kutti R Vinothkumar  2 , Judy Hirst  3
Affiliations

Structure of the Deactive State of Mammalian Respiratory Complex I

James N Blaza et al. Structure. .

Abstract

Complex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, active complex I gradually enters a pronounced resting or deactive state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly active preparation of Bos taurus complex I into the biochemically defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 Å resolution. We show that the deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding to the NADH-reduced enzyme templates their reordering. Our structure both rationalizes biochemical data on the deactive state and offers new insights into its physiological and cellular roles.

Keywords: NADH:ubiquinone oxidoreductase; PEGylated gold grid; cryo-EM; disordered protein structure; electron transport chain; membrane protein; mitochondria.

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Figures

None
Graphical abstract
Figure 1
Figure 1
Spectrophotometric Catalytic Activity Assay of NADH:Decylubiquinone Oxidoreduction by Isolated Deactive Complex I Assay traces comparing enzyme that had been treated by 4 mM NEM (red) with enzyme that had not been treated (green). Without the NEM treatment the deactive protein gradually reactivates, reaching its maximal rate after 150 s. The NEM treatment prevents reactivation and the background rate is only from the small proportion of active enzyme present. Experiments were carried out using 200 μM NADH, 200 μM decylubiquinone, and 0.5 μg mL−1 complex I, as described in the STAR Methods.
Figure 2
Figure 2
Comparison of the Number of Particles Observed Per Micrograph Using PEGylated Gold and Quantifoil Holey Carbon Grids The samples of deactive complex I used were at concentrations of 4.4 mg mL−1 (PEGylated gold UltrAuFoil 0.6/1) and 4.2 mg mL−1 (Quantifoil 0.6/1). The PEGylated gold grids were prepared using a Vitrobot (see STAR Methods) and the Quantifoil grids by manual blotting as described previously (Vinothkumar et al., 2014, Zhu et al., 2016). The data are from two automated data collection sessions on a Titan Krios microscope (see STAR Methods for imaging parameters) and the particles were picked manually in each case.
Figure 3
Figure 3
Classification and Refinement of the Cryo-EM Density Map for Deactive Complex I The RELION pipeline (Scheres, 2012) was used to process data from the deactive preparation. Following manual particle picking and 2D and 3D classification to discard bad particles, 3D refinement and particle polishing were performed. Subsequently, the particles were classified using an angular sampling up to 0.9° and with the resolution limited to 8 Å (see STAR Methods). All three classes provided were populated. The dominant class refined to 4.1 Å, and a minor class to 7.5 Å. The remaining class is negligible as it contained so few particles.
Figure 4
Figure 4
The Structure of Deactive Complex I Is Characterized by Localized Unfolding (A and B) Structure of intact complex I with an arrow showing the view taken of the ubiquinone-binding region (A). The distal section of the membrane domain (shown in wheat) is not included in (B) and (C). (B) View of the ubiquinone-binding region with the subunits involved shown in color as indicated. (C) Close-up of the ubiquinone-binding region, from the same perspective as in (B) with the ubiquinone-binding channel predicted for the class 2 structure (Zhu et al., 2016) shown in blue. The colors are lighter versions of those used in (B). The areas that become disordered in the deactive state (the loops between TMHs 1 and 2 in ND3, TMHs 5 and 6 in ND1, β1 and β2 in the 49 kDa [NDUFS2] subunit, and parts of the 39 kDa [NDUFS9] subunit) are shown in red. His59 is one of the residues likely to interact with the bound ubiquinone head group; Cys39 is the marker residue for the deactive state. The figure was created by combining 5LC5.PDB for the active enzyme (Zhu et al., 2016) with information about the deactive state described here.
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References

    1. Adams P.D., Afonine P.V., Bunkóczi G., Chen V.B., Davis I.W., Echols N., Headd J.J., Hung L.-W., Kapral G.J., Grosse-Kunstleve R.W. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 2010;66:213–221. - PMC - PubMed
    1. Babot M., Birch A., Labarbuta P., Galkin A. Characterisation of the active/de-active transition of mitochondrial complex I. Biochim. Biophys. Acta. 2014;1837:1083–1092. - PMC - PubMed
    1. Babot M., Labarbuta P., Birch A., Kee S., Fuszard M., Botting C.H., Wittig I., Heide H., Galkin A. ND3, ND1 and 39kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I. Biochim. Biophys. Acta. 2014;1837:929–939. - PMC - PubMed
    1. Baradaran R., Berrisford J.M., Minhas G.S., Sazanov L.A. Crystal structure of the entire respiratory complex I. Nature. 2013;494:443–448. - PMC - PubMed
    1. Blaza J.N., Serreli R., Jones A.J.Y., Mohammed K., Hirst J. Kinetic evidence against partitioning of the ubiquinone pool and the catalytic relevance of respiratory-chain supercomplexes. Proc. Natl. Acad. Sci. USA. 2014;111:15735–15740. - PMC - PubMed

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