Functional asymmetry and electron flow in the bovine respirasome

Surprisingly, the two iron-sulfur domains in the complex III dimer are both resolved in the porcine and ovine respirasome, whereas one of them is disordered in our structure of the bovine complex. Therefore neither of the two other structures shows a mobile and therefore catalytically active Rieske protein, as we observe in the bovine respirasome. Letts et al propose that due to the close proximity between complex IV and the Rieske protein of the outer monomer of complex III, the iron-sulfur domain of the outer complex III monomer cannot undergo the conformational changes required for electron transfer. This is consistent with our findings, although neither structure shows any direct protein-protein contacts that would inhibit domain movements. Letts et al conclude that quinone oxidation is likely to take place in the central complex III monomer. This agrees with our model, which is however based on firm experimental evidence of an unresolved, and hence mobile, iron-sulfur domain. Letts et al also propose that quinone reduction in complex III should be catalyzed by the distal monomer; however the respirasome structures provide no evidence for this. Further studies should clarify the exact transfer path of electrons and quinol substrates through the mammalian respirasome.

The 9 Å structure of the mammalian mitochondrial supercomplex shows a well-defined arrangement of the respiratory chain complexes I, III and IV from the inner mitochondrial membrane. The ordered structure of one of the two iron-sulfur domains in the complex III dimer indicates that, contrary to expectation, one of the two electron transfer pathways in complex III is always active, whereas the other monomer is inactive. The active complex III monomer is in a pivotal position between the quinol binding site of complex I and the cytochrome c binding site of complex IV, and hence for directed electron flow through the respirasome. Far from being a random collection of close-packed respiratory chain complexes, the mitochondrial respirasome has not only a clear role for the long-term stability of its component complexes, in particular complex I, but also in optimizing electron flow in cellular respiration.

Bovine heart mitochondria were prepared by differential centrifugation as described (Krause et al., 2005). Mitochondria were solubilized with 1% (w/v) digitonin (Calbiochem) at a detergent-to-protein ratio of 28:1 (Schäfer et al., 2006) or with 0.11% (w/v) PCC-a-M (Glycon) at a detergent-to-protein ratio of 1:1 by incubation for 1 hr at 4°C. Detergent exchange to amphipol A8-35 and protein purification were performed as described before (Althoff et al., 2011).

Proteins were separated on 3–12% polyacrylamide linear gradient gels by BN-PAGE and 2D BN/BN-PAGE (Wittig et al., 2006). Functionally active supercomplexes were detected by in-gel activity assays for NADH:dehydrogenase and cytochrome c oxidase (Grad and Lemire, 2006; Kuonen et al., 1986). Briefly, for complex I, gels were incubated in buffer containing 100 mM Tris, pH 7.4, 0.5 mM NBT (p-nitrotetrazolium blue) and 100 μM β–NADH for 1 hr. For complex IV, gels were incubated for 10–12 hr in 50 mM sodium phosphate buffer at pH 7.2, 0.5 mg/ml DAB (3,3’diaminobenzidine tetrahydrochloride), 1 mg/ml equine heart cytochrome c and 20 U/ml bovine liver catalase.

3 μl of a respirasome sample were applied to freshly glow discharged Quantifoil R2/2 holey carbon grids (Quantifoil Micro Tools, Germany) that had been pretreated in chloroform for 1–2 hr. The grids were blotted for 10 s at 90% humidity and 10°C in an FEI Vitrobot plunge freezer. Immediately before freezing, 0.6 µl of 1 M KCl were added to avoid protein aggregation. Cryo-EM images were collected on a FEI Tecnai Polara operating at 300 kV aligned as previously described (Mills et al., 2013). The microscope was equipped with a Falcon III direct electron detector. Images were recorded manually at a nominal magnification of 59,000x yielding a pixel size at the specimen of 1.77 Å. The camera system recorded 32 frames/s. Videos were collected for 1.5 s with a total of 46 frames with a calibrated dose of about 1.5 e-/Å2 per frame, at defocus values between −1.3 and −4.3 μm.

A set of 3592 micrographs was collected. Whole-image drift correction of each movie was performed using the algorithm developed by Li (Li et al., 2013). Particles were picked manually using EMAN Boxer (Ludtke et al., 1999), and the micrograph-based CTF was determined using CTFFIND3 (Mindell and Grigorieff, 2003) in the RELION 1.4 workflow (Scheres, 2012). The initial dataset contained 156,519 particle images (288 pixels x 288 pixels). Particles were subjected to two-dimensional reference-free classification in RELION 1.4 (Scheres, 2012). Visual selection of particle classes with interpretable features resulted in a dataset of 137,606 particle images for the first 3D consensus refinement. The earlier low-resolution respirasome map (Althoff et al., 2011) was low-pass filtered to 60 Å and used as an initial model for the 3D refinement in RELION 1.4. Individual frames were B-factor weighted and movements of individual particles were reversed by movie frame correction in RELION 1.4 (Scheres, 2014). The resulting dataset of polished particles was used for 3D classification and the best 3D classes were selected for further processing. UCSF Chimera (Pettersen et al., 2004) was used for visualization of cryo-EM maps and docking of atomic models. Figures were drawn with UCSF Chimera.

The cryo-EM maps were deposited in the Electron Microscopy Data Bank with accession code EMD-4107, EMD-4108 and EM-4109 for the cryo-EM maps of class 1, 2 and 3, respectively and the structure coordinates of the complexes fitted to class 1 were deposited in the Protein Data Bank with accession number 5LUF.

We thank Matteo Allegretti for help with cryo-EM data acquisition and image processing, and Natalie Bärland for help with particle picking. This work was funded by the Max Planck Society and the Cluster of Excellence Frankfurt ‘Macromolecular Complexes’ (DFG Project EXC 115).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Sousa JS,Mills DJ,Vonck J,Kuehlbrandt W,2016,Cryo-EM map of bovine respirasome,http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4107,Publicly available at the EBI Protein Data Bank (accession no: EMD-4107)

Sousa JS,Mills DJ,Vonck J,Kuehlbrandt W,2016,Cryo-EM of bovine respirasome,http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4108,Publicly available at th EBI Protein Data Bank (accession no: EMD-4108)

Sousa JS,Mills DJ,Vonck J,Kuehlbrandt W,2016,Cryo-EM of bovine respirasome,http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4109,Publicly available at the EBI Protein Data Bank (accession no: EMD-4109)

Sousa JS,Mills DJ,Vonck J,Kuehlbrandt W,2016,cryo-EM of bovine respirasome,http://www.rcsb.org/pdb/explore.do?structureId=5LUF,Publicly available at the RCSB Protein Data Bank (accession no: 5LUF)

Vinothkumar KR,Zhu J,Hirst J,2014,Electron cryo-microscopy of bovine Complex I,http://www.rcsb.org/pdb/explore/explore.do?structureId=4UQ8,Publicly available at the EBI Protein Data Bank (accession no: 4UQ8)

Iwata S,Lee JW,Okada K,Lee JK,Iwata M,Rasmussen B,Link TA,Ramaswamy S,Jap BK,1999,CYTOCHROME BC1 COMPLEX FROM BOVINE,http://www.rcsb.org/pdb/explore/explore.do?structureId=1BGY,Publicly available at the RCSB Protein Data Bank (accession no: 1BGY)

Tsukihara T,Aoyama H,Yamashita E,Tomizaki T,Yamaguchi H,Shinzawa-Itoh K,Nakashima R,Yaono R,Yoshikawa S,1996,STRUCTURE OF BOVINE HEART CYTOCHROME C OXIDASE AT THE FULLY OXIDIZED STATE,http://www.rcsb.org/pdb/explore/explore.do?structureId=1OCC,Publicly available at the RCSB Protein Data Bank (accession no: 1OCC)