Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis

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Abstract

In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Qo and Qi sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c1 and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.

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Fig. 1: Biochemical characterization of the respiratory supercomplex.
Fig. 2: Architecture of the M. smegmatis respiratory supercomplex.
Fig. 3: Complex III.
Fig. 4: Complex IV.
Fig. 5: Supercomplex cofactors with distances (in Å), measured center to center for the heme groups.
Fig. 6: Model for electron and proton transfer in one half of the supercomplex.

Data availability

The cryo-EM density map and atomic coordinates have been deposited in the Electron Microscopy Data Bank and Protein Data Bank, under accession codes EMD-0289 and PDB 6HWH, respectively. Source data are available from the corresponding authors upon reasonable request.

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Acknowledgements

The C. glutamicum and M. smegmatis strains were provided by M. Bott (Forschungszentrum Jülich) and B. Kana (University of Witwatersrand), respectively. V. Nandicoori (NII, New Delhi) provided the PST-KT plasmid. We thank S. Graf for help with purification of the C. glutamicum supercomplex, and A. Osyczka and E. Berry for valuable discussions. This work was supported by Knut and Alice Wallenberg Foundation (KAW) grants 2013.0006 (P.B., M.H., and P.Ä.) and 2017.0275 (M.H.), and Canadian Institutes of Health Research grant MOP81294 and the Canada Research Chairs program (J.L.R.). The Mass Spectrometry Based Proteomic Facility (Uppsala University), the TEM facility at Stockholm University, and the cryo-EM facility at the Science for Life Laboratory provided the infrastructure. Titan Krios cryo-EM data were collected at the Toronto High-Resolution High-Throughput Cryo-EM Facility, supported by the Canada Foundation for Innovation and Ontario Research Fund.

Author information

R.G.N., P.Ä., M.H., and P.B. conceived and designed the research. R.G.N., O.F., and D.S. performed protein purification, and biochemical and enzymatic analyses. J.S. collected spectral and kinetic data. R.G.N. and Q.K. performed negative-stain EM analyses. B.W. and R.G.N. performed cryo-EM sample preparation and initial data collection and analysis. S.B. collected the final cryo-EM data and performed image analysis. H.G. performed various calculations with the cryo-EM data. B.W. built the de novo atomic structures. J.L.R. supervised the cryo-EM analysis. P.B., M.H., P.Ä., and J.L.R. interpreted data. P.B., J.L.R., and M.H. wrote the manuscript. B.W., M.H., J.S., and J.L.R. prepared data figures and legends.

Correspondence to John L Rubinstein or Peter Brzezinski or Martin Högbom.

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Integrated supplementary information

Supplementary Figure 1 Purification of the intact supercomplex (SC).

a. The SC was purified via a 3×FLAG tag on QcrB. b. Purification profile on 4-16% gradient SDS PAGE. Protein bands were identified by mass spectrometry as indicated. The bands marked by an asterisk (*) showed lower MS scores. Markers (kDa) are indicated. c. SC purified via the 3×FLAG-tag was subjected to SEC. The sample is highly homogenous as evident by the single peak. The elution volumes for the SEC standards Thyroglobulin (669 kDa), Apoferritin (443 kDa) and Amylase (200 kDa) were used to estimate the size of the SC (~970 kDa), as shown in the inset. d. 4-16 % gradient SDS-PAGE after SEC. e. SEC peak fractions were pooled and analyzed with BN-PAGE. Native markers are indicated. f. In order to obtain highly pure sample for mass spectrometry the BN-PAGE band was cut out and run on an SDS-PAGE. Novel components identified by MS are indicated on the left. The bands marked with an asterisk (*) showed lower scores. g. Pyridine hemochrome assay of the supercomplex, reduced minus oxidized spectrum is shown. The peaks of the corresponding hemes are indicated in the figure. The estimated ratio of heme a, b and c is 1.1:0.9:1.3.

Supplementary Figure 2 Composition of the M. smegmatis supercomplex.

In addition to complexes III2 and IV2, four additional subunits are identified based on mass spectrometry. Open reading frame as well as number of predicted transmembrane helices (TMH) (Protter) are given.

Supplementary Figure 3 Cryo-EM map validation.

a. Example micrograph with typical particle images circled in red. b. Example 2-D class average images showing different views of the structure. c. Fourier shell correlation curve for estimation of overall map resolution. d. Estimation of local resolution.

Supplementary Figure 4 M. smegmatis complex III.

Comparison of M. smegmatis complex III quinone binding sites (blue) to those of complex III from S. cerevisiae (3CX5) (green) and chicken (3l71) (purple). a. Qo-site in relation to inhibitor bound structures, purple: 3l71 with azoxystrobin, green, 3cx5 with stigmatellin. MQ9 binds in a pocket formed partly by QcrC (yellow), which has no direct counterpart in the other complex III structures. Structural differences in the α-helix between the respective binding sites highlighted. b. Qi-site in relation to quinone binding in 3l71. Structural differences are highlighted.

Supplementary Figure 5 M. smegmatis cytochrome c oxidase.

a. Cardiolipin-mediated interactions between CtaE and CtaF, bound by two arginine residues, as well as CtaF and complex III. b. in the M. smegmatis SC. All cardiolipin headgroups face the N-side of the membrane.

Supplementary Figure 6 Detection of SodC associated with the III2IV2 supercomplex.

a. The M. smegmatis supercomplex and control protein (left panel) were resolved on CN-PAGE. The bleached band corresponding to the size of the MsSC (“SodC in SC”) indicates SodC activity, not observed in the control protein. A second bleached band (indicated by “free SodC”) running below 66 kDa presumably represents unbound SOD. The right panel shows the same gel after CBB staining to visualize the proteins. b. Inhibition curve for bovine Cu-Zn SOD (Sigma) and the M. smegmatis supercomplex. The concentration of the supercomplex is expressed in pmol of heme a. c. Detection of SodC activity in the M. smegmatis but not the C. glutamicum SC. Left panel- MsSC shows associated positive SodC activity, which is absent in CgSC. Right panel, MsSC, and CgSC protein profile on BN-PAGE.

Supplementary Figure 7 Additional subunits in M. smegmatis cytochrome c oxidase.

a. New subunits identified at complex IV include MSMEG_4692, MSMEG_4693 as well as three unidentified additional subunits. b. Superposition of MSMEG_4692 (blue) to the structure of a TPM-domain with ATPase/ADPase activity from Bizionia argentinensis (5ANP, pink). Despite no detectable sequence homology the domains are structurally very similar with an RMSD of 1.8 Å over 110 aligned residues. TMP-domains are a poorly characterized family shown to be capable of phosphate hydrolysis from various substrates, including nucleotides.

Supplementary Figure 8 Rescue of the M. smegmatis ∆qcrCAB strain by the qcrCAB operon in the plasmid and verification of expression.

a. Colonies of qcrCAB strain harboring the construct PST-KTqcrCAB grow significantly larger than those of the control strain (PST-KT). The 7H9 agar plates are shown after 5 days of growth at 37 °C. b. 7H9 liquid culture. PST-KTqcrCAB bearing cells show comparable growth to wild-type M. smegmatis (bearing PST-KT) while control plasmid (PST-KT) bearing ∆qcrCAB cells show impaired growth. The OD600 after 48 hr of growth at 37°C (starting OD = 0.05) are shown. Error bars indicate SD (n=3). c. Verification of the expression of SC proteins. Detection of FLAG tagged cytochrome b in Ms∆qcrCAB+PST-KTqcrCAB strain by western blotting. A single band is detected at 57 kDa. Lanes 1 and 2 are loaded with lysates from Ms∆qcrCAB+PST-KTqcrCAB and WT+PST-KT cells, respectively. d. Detection of cytochrome cc by heme staining. Membranes purified from the ∆qcrCAB+PST-KTqcrCAB were resolved on SDS-PAGE and subjected to heme staining. The molecular weight of marker proteins is shown on the left.

Supplementary Figure 9 Cryo-EM image processing workflow.

a. Summary of image processing steps for final cryo-EM map generation. b. Orientation distribution plot for particle images contributing to the final cryo-EM map.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9

Reporting Summary

Supplementary Video 1

M. smegmatis supercomplex dynamics. Dynamics of the cyt c domain of the M. smegmatis supercomplex

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