Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance

Abstract Respiratory chains are crucial for cellular energy conversion and consist of multi‐subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high‐resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochrome c between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms.

30th Jun 2020 1st Editorial Decision Dear Prof. Ott, Thank you for the submission of your research manuscript to our journal, which was now seen by three referees, whose reports are copied below.
We concur with the referees that the presented structural analysis and the findings revealing the functional role of supercomplex are very interesting. However, the minor concerns raised by the referees need to be addressed for publication here.
I find the reports informed and constructive, and believe that addressing the concerns raised will significantly strengthen the manuscript. Considering the amount of work required to address these concerns, we believe that three weeks should be sufficient to revise the manuscript. Please let me know if you anticipate problems meeting this deadline.
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Yours sincerely, Deniz Senyilmaz Tiebe Deniz Senyilmaz Tiebe, PhD Editor EMBO Reports Referee #1: The manuscript addresses the highly controversial topic of the physiological role of respiratory supercomplexes. The authors were able to obtain a high resolution structural analysis of the yeast mitochondrial respiratory supercomplex. They identified key residues in the interaction interface and generated site-specific mutants that disrupt supercomplex formation but do not disturb the function of the individual respiratory complexes. By this elegant approach, they were able to obtain unprecedented insight in the functional role of respiratory supercomplexes. They demonstrate that the efficient diffusion of cytochrome c between complexes III and IV represents a crucial function of supercomplexes.
The data is of excellent quality and the manuscript is written in a clear and concise style. The paper will represent a milestone in understanding the structure-function organization of the mitochondrial respiratory chain.
Points to be addressed: 1. The title sounds quite general. In case space permits, it would be very good to include: diffusion of cytochrome c 2. The authors discuss shortly the various controversial models that have been proposed for the role of respiratory supercomplexes, yet due to space limitations they cannot explain the models for the general readership. I suggest to include an additional Table that presents the various models and their suggestions/conclusions and open questions/problems and includes the implications of this study.

Referee #2:
This is an interesting article about the possible mechanistic role of so-called supercomplex arrangement of the catalytic complexes of the respiratory chain. The study makes excellent use of the X-ray structures of the yeast supercomplex (III)2IV, i.e. between a dimer of the cytochrome bc1 complex and a monomer of cytochrome c oxidase. After identifying key residues for making the necessary contact to form the supercomplex, careful mutations of such residues are done to yield a new situation where apparently the complexes are still present in normal amounts but no longer form supercomplexes. Assays at different levels of organisation suggests that electron transfer from NADH to O2, or from succinate to O2 is reduced in the mutant suggesting that the supercomplex is important for assuring maximum efficiency electron transfer between complexes III and IV, via cyt c. There is one problem with the spectroscopy data. Materials and Methods describes methods to assess the contents of cytochrome aa3, heme b, and heme c1 from the samples. However, no mention is given of determining the content of cyt c. The method of determining hemes b and c1 was designed for a pure bc1 complex, yet Table EV2 gives values of "heme c". It is nevertheless clear that cyt c is included based on the given heme contents, which should yield a content of heme c1 of only half the content of heme b. It is important to determine the cyt c content separately, not least to control that the cyt c concentration has not been diminished in the mutant, thus causing the reduced activity. Table EV2 gives the concentrations in uM, but there is no reference to the amount of protein in the different samples. One might of course infer that the content of heme c1 is half that measured for heme b, and subtract that from the results using the method described to yield a relative estimate of cyt c content. However, that is unlikely to yield the correct absolute amount of cyt c due to the methodology. Finally, a more general question. Most mitochondria contain larger amounts of complex IV than complex III, the commonplace number is two-fold. In this work it seems to be about 1.5 fold. This means that with the (III)2IV stoichiometry of the supercomplex, there will be 2-3 times more "free" complex IV units in the membrane than supercomplex units. A comment on how this would be in accordance with the present conclusions would be interesting.

Referee #3:
Respiratory chain complexes play a pivotal role for the energy metabolism. In mitochondria from baker´s yeast, complex III and complex IV associate in supercomplexes. However, the function of the supercomplexes remains unclear. To analyze the role of the supercomplexes, Berndtsson and colleagues used yeast mutant strains with disrupted supercomplexes. Based on their structural model, the authors identified a binding site between Cor1 and Cox5a. Screening various Cor1 variants, they identified a mutant, termed Cor1**, in which the supercomplexes are largely dissociated, but the levels on the individual complexes remain comparable to wild-type mitochondria. Using this mutant strain and an elegant set of biochemical assays, the authors found that formation of respiratory chain supercomplexes promotes the transfer of electrons via cytochrome c and thereby promotes competitive fitness. Overall, the study is well written and the experimental data are of high quality. The conclusions are well-based on experimental findings. I have only minor recommendations for the revision.
The authors show that the respiratory supercomplexes are destabilized in the Cor1** mutants. However, in Figure 1A a small amount of respiratory chain supercomplexes is still detectable using the Cox1 antibody. The authors should adjust their conclusions accordingly (Page 4, first paragraph, last sentence). Alternatively, the authors may also perform pulldowns to verify that complex III and complex IV are dissociated.
We would like to thank the three reviewers for their work with our manuscript, and the very positive evaluation of the work. The three referees raised a number of points that helped us to prepare an improved version. Please find below a point-by-point description of how we implemented their suggestions.

Referee #1:
The manuscript addresses the highly controversial topic of the physiological role of respiratory supercomplexes. The authors were able to obtain a high resolution structural analysis of the yeast mitochondrial respiratory supercomplex. They identified key residues in the interaction interface and generated site-specific mutants that disrupt supercomplex formation but do not disturb the function of the individual respiratory complexes. By this elegant approach, they were able to obtain unprecedented insight in the functional role of respiratory supercomplexes. They demonstrate that the efficient diffusion of cytochrome c between complexes III and IV represents a crucial function of supercomplexes. The data is of excellent quality and the manuscript is written in a clear and concise style. The paper will represent a milestone in understanding the structure-function organization of the mitochondrial respiratory chain. Points to be addressed: 1. The title sounds quite general. In case space permits, it would be very good to include: diffusion of cytochrome c We thank the referee for this comment and changed the title to: "Respiratory supercomplex formation enhances electron transport via cytochrome c diffusion".
2. The authors discuss shortly the various controversial models that have been proposed for the role of respiratory supercomplexes, yet due to space limitations they cannot explain the models for the general readership. I suggest to include an additional Table that presents the various models and their suggestions/conclusions and open questions/problems and includes the implications of this study.
We agree with the referee that the very brief explanation of the models regarding the role of respiratory supercomplexes might be too succinct, especially for the general readership. Since space limitations permitted an extension of the text, we decided to describe the models in more detail in the introduction.

Referee #2:
This is an interesting article about the possible mechanistic role of so-called supercomplex arrangement of the catalytic complexes of the respiratory chain. The study makes excellent use of the X-ray structures of the yeast supercomplex (III)2IV, i.e. between a dimer of the cytochrome bc1 complex and a monomer of cytochrome c oxidase. After identifying key residues for making the necessary contact to form the supercomplex, careful mutations of such residues are done to yield a new situation where apparently the complexes are still present in normal amounts but no longer form supercomplexes. Assays at different levels of organisation suggests that electron transfer from NADH to O2, or from succinate to O2 is reduced in the mutant suggesting that the supercomplex is important for assuring maximum efficiency electron transfer between complexes III and IV, via cyt c.
There is one problem with the spectroscopy data. Materials and Methods describes methods to assess the contents of cytochrome aa3, heme b, and heme c1 from the samples. However, no mention is given of determining the content of cyt c. The method of determining hemes b and c1 was designed for a pure bc1 complex, yet Table EV2 gives values of "heme c". It is nevertheless clear that cyt c is included based on the given heme contents, which should yield a content of heme c1 of only half the content of heme b. It is important to determine the cyt c content separately, not least to control that the cyt c concentration has not been diminished in the mutant, thus causing the reduced activity. Table EV2 gives the concentrations in uM, but there is no reference to the amount of protein in the different samples. One might of course infer that the content of heme c1 is half that measured for heme b, and subtract that from the results using the method described to yield a relative estimate of cyt c content. However, that is unlikely to yield the correct absolute amount of cyt c due to the methodology.
We thank the referee for this critical comment. To clarify that the given heme c content includes both heme c and heme c 1 , we now relabeled this quantification of ctype hemes to "heme cc 1 " in both the table and the respective material and method sections. We agree that it is important to analyze the heme c content separately from heme c 1 , thus we performed additional experiments. We generated mitoplasts via hypotonic treatment to release cytochrome c and separated it from mitoplasts by centrifugation. Performing spectroscopic analyses from the supernatant allowed us to quantify heme c individually and revealed that the Cor1 ** mutant had heme c levels comparable to wild type cells, which is in line with unchanged cytochrome c protein levels presented in Fig. 2 D. Hence, we can exclude that observed phenotypes for the Cor1 ** mutant are due to diminished heme c or reduced cytochrome c protein levels. We added a description of these phenotypes in the respective result section and updated the material and method section accordingly. We apologize for the confusion regarding the given concentrations/missing amount of protein in the samples. For every sample, 200 µg of protein (or the corresponding volume of supernatant in case of heme c quantification) was used. This was only briefly stated in the material and method section. To present this information more clearly, we now also adapted the header of table EV2 accordingly.
Finally, a more general question. Most mitochondria contain larger amounts of complex IV than complex III, the commonplace number is two-fold. In this work it seems to be about 1.5 fold. This means that with the (III)2IV stoichiometry of the supercomplex, there will be 2-3 times more "free" complex IV units in the membrane than supercomplex units. A comment on how this would be in accordance with the present conclusions would be interesting.
In yeast mitochondria supercomplexes exist in two different stoichiometries, namely CIII 2 CIV, and CIII 2 CIV 2 .Therefore most of the CIV and CIII exist in supercomplexes, which is also evident from the Western blot analysis presented in figure 2A. It is therefore highly likely that the organization of the respiratory chain in supercomplexes conveys a substantial advantage for electron transport. The minor fraction of free CIV is therefore expected not to play a major role for the efficiency of OXPHOS, but this will require further experiments to address properly.

Referee #3:
Respiratory chain complexes play a pivotal role for the energy metabolism. In mitochondria from baker´s yeast, complex III and complex IV associate in supercomplexes. However, the function of the supercomplexes remains unclear. To analyze the role of the supercomplexes, Berndtsson and colleagues used yeast mutant strains with disrupted supercomplexes. Based on their structural model, the authors identified a binding site between Cor1 and Cox5a. Screening various Cor1 variants, they identified a mutant, termed Cor1**, in which the supercomplexes are largely dissociated, but the levels on the individual complexes remain comparable to wild-type mitochondria. Using this mutant strain and an elegant set of biochemical assays, the authors found that formation of respiratory chain supercomplexes promotes the transfer of electrons via cytochrome c and thereby promotes competitive fitness. Overall, the study is well written and the experimental data are of high quality. The conclusions are well-based on experimental findings. I have only minor recommendations for the revision.
The authors show that the respiratory supercomplexes are destabilized in the Cor1** mutants. However, in Figure 1A a small amount of respiratory chain supercomplexes is still detectable using the Cox1 antibody. The authors should adjust their conclusions accordingly (Page 4, first paragraph, last sentence). Alternatively, the authors may also perform pulldowns to verify that complex III and complex IV are dissociated.
We thank the referee for this critical comment. As no corresponding signal for CIII in immunoblots probed with the anti Cor1 antibody can be detected, it can be assumed that supercomplexes are entirely destabilized, which also correlates with presented Coomassie stained gels. Thus, we rephrased the mentioned paragraph/sentence as follows: Two of these mutants, namely Cor1 N63A, N187A, D192A (hereafter Cor1 * ), and Cor1 N63A, N187A, D192A, Y65A, V189A, L238A, K240A (Cor1 ** ), lacked higher molecular weight complexes containing both CIII and CIV, thus revealing complete SC disruption (Fig. 1 D).
The authors showed that addition of cytochrome c rescues NADH oxidation in mutant mitoplasts ( Figure 4F). Based on this finding the authors conclude that supercomplex formation facilitates electron transfer from complex III to complex IV via cytochrome c. The conclusion is reasonable. Can the authors provide any experimental data to support this model in intact mitochondria or in cells? For instance, does overexpression of cytochrome c restore the competitive fitness of the mutant cells?
We thank the referee for this excellent suggestion that we have addressed experimentally. We overexpressed cytochrome c to monitor a potential restoration of NADH driven respiration in isolated mitochondria and competitive fitness of the Cor1** mutant. Despite the fact that cytochrome c is described to be a pro-apoptotic protein in both yeast and mammals, our analysis revealed that no increase in cell death nor growth retardation was caused by increased levels of cytochrome c within the first 24 hours after inoculation. Importantly increased levels of cytochrome c corrected the decreased NADH driven respiration in supercomplex-lacking mitochondria. Moreover, overexpression of cytochrome c restored competitive fitness of the Cor1 ** mutant. These results now strengthen our hypothesis that supercomplexes determine competitive fitness via enhancing the efficiency of electron transfer via cytochrome c.
31st Aug 2020 1st Revision -Editorial Decision Dear Martin, Thank you for submitting the revised version of your manuscript. It has now been seen by one of the original referees.
As you can see, the referee finds that the study is significantly improved during revision and recommends publication. Before I can accept the manuscript, I need you to address some minor points below: • We noticed that the reference format should be corrected as follows: where there are more than 10 authors on a paper, 10 will be listed, followed by 'et al.'. Please see https://www.embopress.org/page/journal/14693178/authorguide#referencesformat for more details.
• Please make the data mentioned in he Data Availability section publicly available (CIII2/CIV (EMD-10847 and 6YMX) and CIV model (EMD-10848 and 6YMY)).
Thank you again for giving us to consider your manuscript for EMBO Reports, I look forward to your minor revision.
Kind regards, Deniz --Deniz Senyilmaz Tiebe, PhD Editor EMBO Reports Referee #3: The authors fully addressed all my concerns in the revised version. The new data nicley support the conclusions drawn by the authors. The manuscript provides a highy interesting findings for the role respiratory chain supercomplexes. I strongly recommend publication of this manuscript in EMBO rep..

2nd Sep 2020 2nd Authors' Response to Reviewers
The authors have addressed all minor editorial issues.

10th Sep 2020 2nd Revision -Editorial Decision
Dear Martin, Thank you for submitting your revised manuscript. I have now looked at everything and all is fine. Therefore I am very pleased to accept your manuscript for publication in EMBO Reports.
Congratulations on a nice study! Kind regards, Deniz --Deniz Senyilmaz Tiebe, PhD Editor EMBO Reports At the end of this email I include important information about how to proceed. Please ensure that you take the time to read the information and complete and return the necessary forms to allow us to publish your manuscript as quickly as possible.
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Is there an estimate of variation within each group of data? NA Cor1* mutants were excluded from further analysis after experiments presented in Figure 2, as this mutant displayed altered accumulation of CIII subunits. Since Cor1** also disrupted supercomplexes and did not impair protein levels of CIII or CIV subunits, we analysed this strain to selectively evaluate physiological consequences of supercomplex disruption. Cardiolipin synthase knockout strains were not used for experiments conducted after those shown in Figure 2, since Cor1** was sufficient to disrupt supercomplex formation and cardiolipin synthase knockouts per se had no effect on supercomplex formation. Respective data leading to these decisions are presented in Figure 2, and are described and discussed in the manuscript. Outliers were defined as data points outside the 2.2-fold interquartile range (IQR) and are highlighted in turquoise. Upon presence of outliers, alternative non-parametric tests were performed (described in detail in Table  EV6). Of note, outliers were not excluded from the analysis.
No randomization was performed. Covariates were excluded by using isogenic yeast strains, independent clones to exclude clonogenic variations, simultaneous inoculation of all strains within an experiment to equal optical density, using the same batch of media. In competitive fitness analyses, different selection markers had to be used for wild type cells and Cor1 mutants. Hence, control experiments were performed with switched selection markers and results are presented in the manuscript.

Manuscript Number: EMBOR-2020-51015V1
The number of n given for each experiment represents biological replicates. Thereby, experiments were performed with several clones (at least 3 per genotype) to exclude clonogenic variations or with mitochondria obtained from individual preparations (at least 3 per strain). A detailed description of statistical analysis is given in the method section.
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