Review
The functional significance of mitochondrial
respiratory chain supercomplexes
Andreas Kohler1,2 , Antoni Barrientos3,4
Abstract
The mitochondrial respiratory chain (MRC) is a key energy trans-
ducer in eukaryotic cells. Four respiratory chain complexes cooper-
ate in the transfer of electrons derived from various metabolic
pathways to molecular oxygen, thereby establishing an electro-
chemical gradient over the inner mitochondrial membrane that
powers ATP synthesis. This electron transport relies on mobile elec-
tron carries that functionally connect the complexes. While the
individual complexes can operate independently, they are in situ
organized into large assemblies termed respiratory supercom-
plexes. Recent structural and functional studies have provided
some answers to the question of whether the supercomplex orga-
nization confers an advantage for cellular energy conversion. How-
ever, the jury is still out, regarding the universality of these claims.
In this review, we discuss the current knowledge on the functional
significance of MRC supercomplexes, highlight experimental limi-
tations, and suggest potential new strategies to overcome these
obstacles.
Keywords bioenergetics; electron transfer; Mitochondria; respiratory chain;
supercomplexes
Subject Categories Metabolism; Organelles
DOI 10.15252/embr.202357092 | Received 28 February 2023 | Revised 10 July
2023 | Accepted 14 September 2023 | Published online 12 October 2023
EMBO Reports (2023) 24: e57092
The mitochondrial respiratory chain
As the most versatile form of chemical energy in cells, ATP is syn-
thesized in large quantities by mitochondrial activity. Biochemical
reactions, many occurring in the cytoplasm, convert carbohydrates
and lipids to mitochondrial acetyl-CoA. This central metabolite is
processed in the mitochondrial matrix by the tricarboxylic acid cycle
(TCA; Fig 1). Electrons originating from these TCA reactions are
passed over to the typically four multiprotein complexes (CI-CIV) of
the electron transport chain. In a process termed oxidative phos-
phorylation (OXPHOS), the electrons are transported through the
1 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
2 Institute of Molecular Biosciences, University of Graz, Graz, Austria
3 Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA
4 Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, FL, USA
5 Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden
*Corresponding author. Tel: +46 31 786 9402; E-mail: martin.ott@gu.se
ª 2023 The Authors. Published under the terms of the CC BY 4.0 license.
, Flavia Fontanesi4 & Martin Ott1,5,*
mitochondrial respiratory chain (MRC), where they finally reduce
molecular oxygen to form water (Fig 1). The energy of this reaction
is converted into an electrochemical gradient across the inner mito-
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chondrial membrane (IMM). In a subsequent step, protons flow
from the mitochondrial intermembrane space (IMS) back into the
matrix, powering the ATP synthase (CV), which condenses ADP
and phosphate to form ATP (Fig 1).
NADH feeds two electrons into the NADH dehydrogenase (CI),
the first and largest of the MRC multiprotein complexes in mam-
mals, consisting of 45 subunits and comprising a mass of approx.
1 MDa (Kampjut & Sazanov, 2020; Fig 1). Electrons from NADH are
thereby transferred to oxidized quinone (Q), a reaction coupled to
proton extrusion from the matrix into the IMS via CI’s antiporter
subunits (Jones et al, 2017; Fig 1). Q is an abundant mitochondrial
lipid that can be reversibly reduced and oxidized, respectively, and
freely diffuses in the IMM. Interestingly, some species, including
the model organism Saccharomyces cerevisiae, do not contain a con-
ventional CI, but rather possess non-proton-translocating peripheral
membrane NADH dehydrogenases (Nde1, Nde2, and Ndi1; Joseph-
Horne et al, 2001). In addition, electrons extracted from succinate
can reduce Q via the succinate dehydrogenase (CII; Fig 1). Whereas
CI pumps protons, no H+ is translocated by CII. Of note, Q can also
be reduced by several other membrane-bound dehydrogenases
(e.g., glycerol 3-phosphate dehydrogenase). The reduced quinone
(QH2) subsequently transfers two electrons to cytochrome bc1
reductase (CIII), which exists as an obligate homodimer (CIII2;
Fig 1) in all species (Stephan & Ott, 2020). CIII couples electron
transfer from QH2 to cytochrome c (Cytc) with the shuttling of H+
into the IMS via a mechanism called Q-cycle (Mitchell, 1975;
Osyczka et al, 2005). In one such Q-cycle, two QH2 molecules are
oxidized to Q, releasing 4 H+ into the IMS and reducing two Cytc
molecules (Fig 1). Of note, another Q is reduced to QH2 in this pro-
cess, which can subsequently be used for another round of the Q-
cycle. The protein Cytc acts as a mobile electron carrier, with a cen-
tral, covalently attached heme group that can reversibly bind one
electron. Cytc transfers electrons from CIII to Cytc oxidase (CIV),
where molecular oxygen is reduced to water (Fig 1). In this last step
of the electron transport chain, another 4 H+ are pumped into the
IMS by oxidizing 4 Cytc molecules (Wikstrom, 1977). Finally, the
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EMBO reports Andreas Kohler et al

Glycolysis β-oxidation

Acetyl CoA

Citrate
NADH Oxaloacetate cis-Aconitate

NAD+
Isocitrate
Malate Tricarboxylic acid cycle NAD+
TCA
(TCA) NADH
Fumarate α-Ketoglutarate
FADH2
NAD+
FAD
Succinate
Succinyl CoA
NADH

GTP GDP

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OMM

CI CII CIII CIV

NADH-ubiquinone Succinate-ubiquinone Ubiquinol-cytochrome c e- cytochrome c CV
oxidoreductase oxidoreductase oxidoreductase oxidase ATPsynthase
IMS
4H+ 4H+ 2H+
cytochrome c

QH2 QH2 2Q
IMM
Q Q 2QH2
2H+ 2H+ 2.7 H+
1/2 O2
4H+
H 2O
4H+
Succinate Fumarate +
+ 2H+ 2H

NAD++ H+ ADP + Pi
NADH ATP

Figure 1. The tricarboxylic acid cycle (TCA) and mitochondrial respiratory chain.
Upper Panel: Acetyl-CoA is a central metabolite produced via various metabolic reaction pathways. It can subsequently be utilized in the TCA, were among others sev-
eral molecules of NADH and FADH2 are generated. Lower Panel: Electrons from these metabolites can be subsequently utilized by the mitochondrial respiratory chain
to convert energy in the form of ATP. Structures of the individual respiratory chain complexes and the electron carrier cytochrome c are shown, as well as respective
redox reactions (PDB CI: 6ZKC; PDB CII: 1ZP0; PDB CIII: 6Q9E; PDB CIV and CytC: 5IY5; PDB CV: 5ARA). IMM, inner mitochondrial membrane; IMS, intermembrane space;
OMM, outer mitochondrial membrane.

electrochemical gradient across the IMM formed by the transfer of
protons from the mitochondrial matrix into the IMS is used by the
ATP synthase (CV), which phosphorylates ADP with inorganic
phosphate to ATP, whereby the flow of protons back into the
matrix powers ATP synthesis (Mitchell, 1975) by a rotary mecha-
nism of the ATP synthase (Fig 1).
Connectivity of mitochondrial respiratory chain complexes
Despite detailed insights into the fundamental structural and cata-
lytic properties of the individual MRC complexes, available since
last century, their overall structural organization and physiological
implications of that organization have been extensively revisited
over the last 20 years and are still debated today (Milenkovic et al,
2017). Historically, two opposing models for how the complexes are
functionally connected were proposed early on, namely, the “solid-
state” and the “liquid-state” (also referred to as “fluid-state”)
models. The solid-state model postulates that all components of the
MRC (including Q and Cytc) form a single unit, which can catalyze
the entire process of mitochondrial respiration. Thereby, Q and Cytc
mediate electron transfer via enclosed pathways, never exchanging
with the exterior of the unit (Ragan & Heron, 1978). On the con-
trary, the liquid-state model proposes that individual complexes
reside independently in the IMM. Likewise, Q and Cytc diffuse freely
and randomly to transfer electrons between the individual com-
plexes (Hochli & Hackenbrock, 1976; Hackenbrock et al, 1986). The
liquid-state model received extensive experimental support, mostly
owing to the observation that individual MRC complexes could be
purified in active forms and that the small mobile electron carriers
are freely diffusing and in large stoichiometric excess over MRC
complexes. However, the solid-state model gained new experimen-
tal support. Through the use of mild detergents for mitochondrial
membrane solubilization and the establishment of blue native

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Andreas Kohler et al EMBO reports

polyacrylamide gel electrophoresis, it was demonstrated that MRC
complexes form supramolecular assemblies, termed respiratory
chain supercomplexes (SCs) (Cruciat et al, 2000; Sch€ agger &
Pfeiffer, 2000). SCs were shown to exist in varying stoichiometry
in different species, ranging from bacteria to plants, fungi, and
animals. Because they exist widely in multiple life forms, they
must confer specific, selectable advantages to organisms. In this
review, we provide a comprehensive overview of the various
forms of mitochondrial SCs, their biogenesis, and their structure
and discuss the current understanding of the functional roles
of SCs.
Structures and assembly of different respiratory chain
complexes
Supercomplexes are also formed between CIII and CIV without
CI, with the stoichiometries CIII2 + CIV2 or CIII2 + CIV, respectively
(Fig 2A). Whereas CIII forms an obligate homodimer (CIII2), CIV
can occur in its monomeric, as well as in its dimeric (CIV2) form.
The respirasomes
A common feature of respirasomes is the curving of the membrane
arm of CI around the CIII dimer, which can already be observed in
the isolated CI + CIII2 complex (Letts et al, 2019). The overall struc-
ture of these SCs was similar to the respirasome configuration, but
showed at least six open states (inactive form) of CI and one closed
state (active form) (Letts et al, 2019). In structural studies for both
ovine and bovine respirasomes, two different classes had significant
variations of the CIV position (Letts et al, 2016; Sousa et al, 2016).
For the ovine respirasome structure, “loose” (active) and “tight”

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Overview of different supercomplex configurations
Only a small fraction of CI is present as a free complex in bovine
heart mitochondria, and in some studies using cell cultures, the free
form of CI is even undetectable (Lobo-Jarne et al, 2018), whereas
the rest is found in various SC assemblies with varying stoichiome-
try (Sch€agger & Pfeiffer, 2001) (Fig 2A). The most prevalent of these
assemblies in human cells is the respirasome, consisting of
CI + CIII2 + CIV and a size of approx. 1.7 MDa, accounting for
approximately half of CI in SCs (Sch€agger & Pfeiffer, 2001; Gu
et al, 2016; Letts et al, 2016; Wu et al, 2016; Lobo-Jarne &
Ugalde, 2018; Fig 2A). Other variants of the respirasome, which
have been proposed to contain further copies of CIV (CI +
CIII2 + CIV2-4), present only minor amounts of CI-containing SCs.
The second most prevalent assembly of CI is the CI + CIII2 configu-
ration (Sch€agger & Pfeiffer, 2001; Fig 2A). Of note, although the stoi-
chiometry of these assemblies is the same between yeasts, plants,
and mammals, the structures and frequencies differ (Davies
et al, 2018). Evidence for the existence of even larger assemblies
than the CI + CIII2 + CIV4 respirasome comes from cryo-EM studies
in HEK293T cells, reporting a symmetrical respiratory megacomplex
with the stoichiometry CI2 + CIII2 + CIV2 (Guo et al, 2017).
Although the authors also speculate that two units of CII could
potentially bind to this megacomplex, a corresponding unassigned
density could not be observed in their data, and so far, no direct
experimental evidence for the participation of CII in supercomplex
assembly of metazoa or plants was reported (Caruana &
Stroud, 2020). However, a recent study using the ciliate protist Tet-
rahymena thermophila reports a CI + CII + CIII2 + CIV2 SC, which
has important implications in the curvature of the IMM in this
organism (Mu € hleip et al, 2023). Of note, this arrangement shows
significant differences compared to known mammalian SCs.
(inactive) conformations were shown, where CIV contacts both CI
and CIII in the tight variant but only CI in the loose form (Letts
et al, 2016). Class 1 of the bovine respirasome is similar to the tight
form and represents the most prevalent population and stable form
of the respirasome (Gu et al, 2016; Letts et al, 2016; Letts &
Sazanov, 2017). Two main interaction sites are found between CI
and CIII2, namely, one in the membrane between NDUFA11 and the
UQCRB, UQCRQ, and UQCRH subunits of CIII, and a second one in
the matrix between NDUFB4, NDUFB9, and CIII subunit UQCRC1
(Fig 2B). Interestingly, the contacts formed between CI and CIII
within the respirasome involve so-called supernumerary subunits.
Compared to the 14 conserved CI subunits of bacterial origin, these
supernumerary subunits are assumed to be of eukaryotic origin
(Gray, 2012). As no CI-containing supercomplexes have been identi-
fied in bacteria so far (however, CIII2 + CIV2 supercomplexes were
described, Kaila & Wikström, 2021), it is feasible that certain CI
supernumerary subunits evolved to form respirasomes (Letts &
Sazanov, 2017). On the contrary, the contacts between CI and CIV
are formed by the evolutionarily conserved core subunit ND5 of CI
and the CIV subunit COX7C (Gu et al, 2016; Letts et al, 2016;
Fig 2B).
Different states of the respirasome can be observed when looking
at the four major rotational axes of its structure, including the hinge
between the matrix and membrane arms of CI, the pivot between CI
and CIII2, the rotation along the two-fold-symmetry axis of CIII2,
and the internal asymmetric rotation of CIV (Letts et al, 2016; Letts
& Sazanov, 2017). Whereas the rotation between the matrix and
membrane arms of CI may be associated with the regulation of
enzymatic activity or may represent different stages in the catalytic
cycle (Letts et al, 2016; Agip et al, 2018; Blaza et al, 2018; Kravchuk
et al, 2022), physiological implications of other conformational
states remain unknown. It might be that these different

Figure 2. Respiratory chain supercomplex assemblies.

(A) Stoichiometry of several mitochondrial respiratory chain supercomplexes. (B) Main interaction surfaces of mammalian respirasomes (CI + CIII2 + CIV, PDB: 5j4z).
Close-up of interactions between CI (green) and CIII (blue) subunits NDUFA11, UQCRB, UQCRQ, and UQCRH (left panel), as well as NDUFB4 and NDUFB9 with UQCRC1
▸
(lower right panel) are shown, as well as the interaction surface between CIII and CIV (red), formed by the CI subunit ND5 and the CIV subunit COX7C (upper right
panel). Visualization is adapted from (Letts et al, 2016). (C) Interaction surfaces of the Saccharomyces cerevisiae respiratory chain supercomplex CIII2 + CIV (PDB: 6YMX).
The inner membrane protein–lipid–protein interaction between Rip1 (CIII, blue, cardiolipin (CL) and Cox5 (CIV, red) are shown (left panel), as well as the matrix-situated
protein–protein interaction between Cor1 and Cox5a). Visualization was modified from (Berndtsson et al, 2020). (D) Structure of the mammalian respiratory chain
supercomplex CIII2 + CIV in the unlocked configuration (PDB: 7O3C, upper panel). The lower panel shows a transparent visualization of the same structure to display
interactions between the CIII (blue) subunit Sub9 and the CIV (red) subunit SCAF1.

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EMBO reports Andreas Kohler et al

A
CI + CIII2 + CIV CI + CIII2 CIII2 + CIV2 CIII2 + CIV

CI Complex I CIII Complex III CIV Complex IV

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COX7C
UQCRB NDUFA11

ND5

UQCRQ NDUFB4 UQCR1

UQCRH

NDUFB9

C D

Qcr8 Cox5

CL
Cor1 SCAF1

Rip1 Cox5

Sub9

Figure 2.

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Andreas Kohler et al
conformations present intermediate states of respirasome assembly
or that some configurations (e.g., the loose form of the respirasome)
are sample preparation artifacts (Letts et al, 2016).
The exact mechanism of respirasome formation, particularly
involving SC assembly factors, is still a matter of debate. The CIV
subunit COX7A has two tissue-specific isoforms, namely, COX7A1
in the heart and skeletal muscles and COX7A2 in the liver. However,
both isoforms can be found in heart-derived mitochondria, whereas
COX7A2 is found in liver mitochondria only. Further, a homolog of
COX7A2, COX7A2L, or SCAF1 was described, initially thought to be
essential for both respirasome and CIII2 + CIV SC formation (Ikeda
et al, 2013; Lapuente-Brun et al, 2013). It was postulated that SCAF1
is required to form respirasomes in all tissues except in the heart
and skeletal muscle (Cogliati et al, 2016). However, overwhelming
evidence shows that SCAF1 is only required for CIII2 + CIV SC for-
mation but not for the respirasome (Davoudi et al, 2016; Jha
et al, 2016; Perez-Perez et al, 2016; Sun et al, 2016; Williams
et al, 2016). Although SCAF1 is essential for the assembly of the
CIII2 + CIV SC (as discussed in more detail below), it does not con-
tribute to the assembly of the respirasome, as the formation of
the CIII2 + CIV SC is not necessary for respirasome formation (Pe-
rez-Perez et al, 2016). Likewise, SCAF1 was not detected in the
structure of the respirasome. However, a recent analysis of human
cells by complexome profiling has led to the identification of two
distinct respirasome forms that present slightly different electropho-
retic mobility and contain either SCAF1 or COX7A2 isoforms (Fern a-
ndez-Vizarra et al, 2022).
Supercomplex CI + CIII2 and CIII2 + CIV
Although the configuration of CI + CIII2 is observed much less fre-
quently in mammalian cells as compared to the respirasome
(approx. 17% of total complex I in mitochondria compared to
approx. 54% for the respirasome) (Sch€agger & Pfeiffer, 2001), this
arrangement is widely observed across kingdoms (Davies
et al, 2018).
Recently, high-resolution structures of T. thermophila respiratory
chain complexes have been solved, including CI + CIII2 (Zhou
et al, 2022). Interestingly, the overall arrangement is similar to that
of mammalian CI + CIII2 SCs as discussed above. However, signifi-
cant differences in the interaction sites were observed, with more
contacts in both the matrix and the IMM, as well as an IMS bridge
present in T. thermophila SCs. Thereby, these interactions cause
structural and functional symmetry breaking in CIII2. The authors
further suggested that no free CI is present in T. thermophila and
that the CI + CIII2 represents the minimal organization of CI (Zhou
et al, 2022). Moreover, very recent cryo-EM structures of Arabi-
dopsis thaliana CI + CIII2 showed a plant-specific additional interac-
tion site between the complexes compared to ovine CI + CIII2
structures, potentially contributing to the unusual stability of the
plant SC (Klusch et al, 2023). Similar plant-specific interaction sur-
face between CI and CIII2 was also shown for Vigna radiata (Maldo-
nado et al, 2023).
In the yeast S. cerevisiae that lacks a canonical CI, the ratio
between the two SC species CIII2 + CIV2 or CIII2 + CIV reflects the
cellular energy demands under different growth conditions
(Sch€agger & Pfeiffer, 2000). In particular, the abundance of SC
CIII2 + CIV2 is enhanced in cells propagating in respiratory
media and is mainly controlled by the amount of CIV available
 2023 The Authors
EMBO reports
(Sch€agger & Pfeiffer, 2000). The first elucidated high-resolution
structure for mitochondrial CIII2 + CIV came from Saccharomyces
cerevisiae (Hartley et al, 2019; Rathore et al, 2019). The association
between CIII2 and CIV occurs via protein–protein interactions
between the CIII subunit Cor1 and the CIV subunit Cox5, stabilizing
the complex from the matrix side, and a protein–lipid–protein inter-
action between Cor1, cardiolipin, and the CIV subunit Rip1 (Fig 2C).
Whereas alanine substitutions in Cor1 abolished the formation of
SCs, the reduction of cardiolipin content via deletion of the cardio-
lipin synthase Crd1 had no direct consequence on SC formation
(Pfeiffer et al, 2003), showing that the protein–protein interaction
between Cor1 and Cox5 is necessary and sufficient for the formation
of CIII2 + CIV SCs in yeast (Berndtsson et al, 2020). However, the
yeast and mammalian CIII2 + CIV differ substantially in their struc-
ture. In yeast, CIV binds to CIII2 at the same site as CI does in the
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respirasome, resulting in different binding modes between CIII and
CIV in yeast and mammals (Rathore et al, 2019). CIII2 + CIV of
mammals was observed in two different conformations, a locked
and an unlocked state (Vercellino & Sazanov, 2021). In the unlocked
state, CIV is peripherally attached to CIII2, and almost 180° rotated
compared to the yeast form of this assembly. Whereas the unlocked
conformation contained both fully mature CIII2 and CIV, the locked
form harbors assembly intermediates of CIII2.
The assembly of mammalian CIII2 + CIV has been shown to fol-
low a specific sequence. The assembly factor SCAF1 binds to a pre-
mature form of CIII2 and drives the docking of CIV to form an
intermediate-locked assembly. This is followed by the maturation of
CIII2, forming the locked conformation. Subsequently, CIV shifts to
the unlocked state, in which the newly added subunits of the mature
CIII2 form contacts with CIV (Vercellino & Sazanov, 2021; Fig 2D).
The yeast proteins Rcf1 (and its mammalian homolog HIGD2A),
as well as Rcf2 (yeast-specific), were previously suggested to medi-
ate assembly and stability of CIII2 + CIV SCs (Chen et al, 2012; Stro-
golova et al, 2012; Vukotic et al, 2012; Zhou et al, 2018). However,
Rcf1 was later shown to interact with CIV, thereby regulating the
late stages of its assembly (Su et al, 2014; Garlich et al, 2017). In
addition, a recent study showed that Rcf1 and Rcf2 regulate respira-
tion of yeast cells, without significantly affecting the assembly of
SCs (Dawitz et al, 2020). It was also shown that Rcf1 and Rcf2 are
not stoichiometric subunits of CIV, and thus, they were also not pre-
sent in the cryo-EM structure of yeast SCs under physiological con-
ditions (Rathore et al, 2019). However, Rcf2 could be detected in SC
structures under hypoxia-mimicking conditions (Hartley et al,
2020). This is in line with the observation that Rcf1 and Rcf2 regu-
late CIV activity, in particular under energy-demanding conditions
(Dawitz et al, 2020). These results suggest that the primary func-
tions of Rcf1 and Rcf2 are related to the assembly of individual com-
plexes and regulating their function rather than specifically
mediating SC formation. In mammals, other relevant HIGD family
proteins are HIGD1A and HIGD1C. HIGD1A impacts the assembly of
CIII (Timon-Gomez et al, 2020), and in addition, it binds to Cytc and
CIV in the respirasome to enhance electron transfer (Hayashi
et al, 2015; Guerra-Castellano et al, 2018). In agreement, HIGD1A
overexpression improves cellular respiration in models of mitochon-
drial disorders (Nagao et al, 2020). Differently, HIGD1C is almost
exclusively expressed in glomus cells, where it also interacts with
CIV to negatively regulate its activity and facilitate oxygen sensing
(Tim on-Gomez et al, 2022).
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Physiological functions of supercomplexes
The physiological functions of SCs remain controversial, despite
more than two decades of research (Sch€agger & Pfeiffer, 2000). Sev-
eral hypotheses regarding their physiological significance were
made and will be discussed in this section.
Free diffusion versus substrate channeling
The random collision model suggests that the electron transport
within mitochondria is a diffusion-based process. The mechanistic
role of Q to transport electrons from NADH or succinate oxidation to
CIII was established in the 1960s, when it was demonstrated that a
surprisingly high concentration of Q was found in the IMM, where it
behaves as a pool, binding electrons (and protons) to the Q head-
group, and thereby helps to connect electron flow in the MRC
(Fig 3A). Because Q is a substantially hydrophobic molecule, owing
to the attachment of varying numbers of isoprenyl moieties (Stefely
& Pagliarini, 2017), the coenzyme is solved in the lipidic core of the
membrane, where it diffuses in the plane of the lipid bilayer. In con-
trast, Cytc is a protein of roughly 15 kDa molecular weight that
carries a covalently attached heme moiety enabling the transport of
one electron at a time. Cytc belongs to the class of peripheral mem-
brane proteins. In the absence of transmembrane segments,
membrane association stems from its attachment to the IMM by elec-
trostatic interactions with negatively charged phospholipids, particu-
larly cardiolipin, the signature phospholipid of mitochondria
(Kinnunen et al, 1994). Moreover, biophysical experiments
suggested that in addition to electrostatic interactions, also an alter-
native form of membrane interaction could exist. In this model, an
acyl chain of cardiolipin might get inserted into the folded Cytc, giv-
ing rise to a tight membrane-bound form (Rytomaa &
Kinnunen, 1995). However, Cytc release from mitochondria during
apoptosis is a highly conserved and important event, necessitating
that the protein is detached from the IMM (Ott et al, 2007). This pro-
pensity of Cytc, namely to interact with the IMM via various mecha-
nisms and the well-documented fact that it can be detached from the
membrane to act as a signaling molecule during cell death execution
(Garrido et al, 2006), complicates the interpretation of how the pro-
tein diffuses inside mitochondria. However, the general consent is
that Cytc is freely moving in the IMS (Hochman et al, 1982), either
moving in two dimensions at the level of the IMM, in three dimen-
sions in the IMS, or a mixture of both, whereby the actual ionic
strength might dictate the degree of membrane association (Fig 3B).
The discovery of SCs, however, led to the hypothesis that
respirasomes might act as solid-state units, with each respirasome
carrying its own Q and Cytc molecules and catalyzing the transfer of
electrons from NADH to O2 (Fig 3A and B). Flux control analyses
take advantage of inhibitors to block a certain step in a reaction
pathway. If the effect on the isolated step is equal to the effect on
Figure 3. Physiological functions of respiratory chain supercomplexes.
(A) Comparison between the free diffusion and substrate channeling model for coenzyme Q (Q) within respiratory chain supercomplexes. Arrows indicate the free
exchange with the Q pool (free diffusion model, left panel) or the direct Q transfer in the form of substrate channeling (right panel). (B) Comparison of various
postulated models for cytochrome c shuttling between complex CIII and CIV in respiratory chain supercomplex configuration. Arrows indicate the modality of
cytochrome c transfer/exchange with the free pool, similar as described for the Q pool above. (C) Potential physiological functions of different respiratory chain
supercomplex assemblies. Several of these assigned functions are controversially discussed. Arrows indicate a respective increase or decrease of the indicated
physiological functions and pathophysiological effects.
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Andreas Kohler et al
the overall pathway (and hence, the flux control coefficient is 1),
this step has complete control over the pathway. Such an analysis
has been performed for the NADH oxidation pathway with the
inhibitors rotenone and mucidin, and it was shown that CI and CIII
behave as a single functioning unit (Blanchi et al, 2004). In support
of this model, Q and Cytc were found in respirasomes and shown to
be able to transfer electrons from NADH to O2 (Acın-Perez
et al, 2008). It was therefore proposed that neither Q nor Cytc form
homogeneous pools, but instead, each respirasome contains a dedi-
cated small pool of Q and Cytc, which are not exchanged and allow
a localized electron transfer via substrate channeling (Lapuente-
Brun et al, 2013; Fig 3A and B). Substrate channeling in complexes
of enzymes that act sequentially in a pathway is a process where a
specific substrate is transferred from one enzymatic activity to the
next without allowing free diffusion of the substrate into the bulk
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solution. For example, the large E1-E2-E3 enzymes like pyruvate
dehydrogenase or alpha-ketoglutarate dehydrogenase employ sub-
strate channeling by transferring a covalently bound substrate
between the complexes’ three enzymatic entities using a highly flex-
ible domain.
Although the substrate channeling hypothesis represents an
attractive model for the physiological functions of SCs, experimental
evidence against this hypothesis emerged. First, repetitions of flux
control experiments showed that the outcome of these experiments
strongly depended on the respective inhibitor used (Blaza
et al, 2014). As certain inhibitors even resulted in a biological mean-
ingless flux control coefficient of > 1, this approach is not a reliable
method to analyze physiological functions of SCs (Blaza et al, 2014;
Milenkovic et al, 2017). The authors also measured substrate oxida-
tion using NADH, succinate, or both in submitochondrial particles
containing supercomplexes, prepared with and without the addition
of Cytc to alter the rate-limiting step for substrate oxidation. The
same system with the same set of supercomplexes showed both
additive and non-additive kinetics (Blaza et al, 2014). These results
reject the hypothesis of a Q pool partitioning in SCs. Similarly, spec-
troscopic measurements in living cells also demonstrated that Cytc
does not face any barriers to free diffusion (Trouillard et al, 2011).
In an additional study, an alternative QH2 oxidase was inserted into
mammalian heart mitochondrial membranes, which then presents a
competing pathway for QH2 oxidation (Fedor & Hirst, 2018). In the
case of substrate channeling, the flux through the competing path-
way should be negligible. However, the rate of cyanide-insensitive
NADH:O2 oxidoreduction via the alternative QH2 oxidase was signif-
icantly higher, revealing that CIII/CIV catalysis is rate-limiting for
both, NADH and succinate oxidation, and that Q is not channeled in
SCs. In addition, structures of the mammalian respirasomes (Gu
et al, 2016; Letts et al, 2016) and CIII2 + CIV SCs (Vercellino &
Sazanov, 2021), yeast CIII2 + CIV1-2 SCs (Hartley et al, 2019;
Rathore et al, 2019; Berndtsson et al, 2020), or plant CIII2 + CIV SCs
▸
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Andreas Kohler et al EMBO reports

A
Free diffusion Substrate channeling
exchange with pool no exchange with pool
CI Complex I

Q Q
Q Q Q Q Q Q CIII Complex III
Q Q
Q Q Q Q
CIV Complex IV
Q
Q Coenzyme Q

Cytochrome c

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B
Free diffusion Substrate channeling 2D diffusion 2D diffusion
exchange with pool no exchange with pool no exchange with pool exchange with pool

C
CI + CIII2 + CIV CI + CIII2 CIII2 + CIV

Oxidative stress Oxidative stress Competitive fitness

Protein aggregation Protein aggregation Protein aggregation
Cooperative assembly Cooperative assembly Cooperative assembly
Stability of complexes Metabolic efficiency

Figure 3.

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EMBO reports
(Maldonado et al, 2021) do not contain barriers restricting diffusion
of Q or Cytc away from the complexes, features that would be neces-
sary for substrate channeling. Direct tunneling of electrons between
CIII2 + CIV in the MRC can also be excluded because of a too-large
distance (> 6 nm) between the respective Cytc binding sites
(Rathore et al, 2019; Vercellino & Sazanov, 2021), allowing electron
transfer only by a mobile pool of Cytc. However, bacterial SCs do
contain covalently bound Cytc, clearly allowing substrate channel-
ing to transfer electrons from CIII to CIV (Daldal et al, 2003; Gong
et al, 2018; Kao et al, 2022).
Efficiency of electron transfer and competitive fitness
While substrate channeling in a strict definition can be excluded as
a possibility to explain the occurrence of SC in mitochondria, it is
still feasible that a bioenergetic advantage of SC formation can
derive from an increased efficiency of electron transfer between the
individual complexes (Fig 3C). One approach to test this hypothesis
would be to delete factors essential for the formation of SCs and
then determine electron transfer efficiencies. This has been
performed for SCAF1, which, as stated above, does not affect the
formation of respirasomes, but the assembly of CIII2 + CIV SCs.
The ablation of SCAF1 did not affect bioenergetics, but rather pro-
voked changes in nutrient sensing (Perez-Perez et al, 2016; Lobo-
Jarne & Ugalde, 2018). In contrast, bioenergetic differences were
observed in a zebrafish model, where absence of SCAF1 caused
abnormal fat deposition and reduction in fertility (Garcıa-Poyatos
et al, 2020). As further discussed below, controversial results were
reported in mouse models, showing either measurable or no effects
on energy metabolism upon deletion of SCAF1 (Lapuente-Brun
et al, 2013; Mourier et al, 2014; Shiba et al, 2017).
Moreover, in many systems, including mammalian mitochon-
dria, the respiratory chain complexes assemble in a variety of super-
complexes with varying degrees of stoichiometries, thereby
complicating analyses of the bioenergetic efficiency of the different
assemblies. Here, yeast offers a unique advantage by lacking CI and
having CIII and CIV organized primarily in common supercom-
plexes. We recently used information from the cryo-EM structure of
S. cerevisiae CIII2 + CIV1-2 (Rathore et al, 2019) to selectively disrupt
SCs without affecting individual complexes. We genetically engi-
neered mutants of Cor1, expressed under their endogenous pro-
moter at physiological levels (Berndtsson et al, 2020). These point
mutations caused the disruption of CIII2 + CIV1-2 SCs as the only SC
species present in S. cerevisiae, without affecting individual com-
plexes. This selective disruption impaired electron transport by
impacting the diffusion efficiency of Cytc between the individual
complexes (Berndtsson et al, 2020). Increased concentrations of
Cytc could correct for this defect. Interestingly, this decreased effi-
ciency of electron transfer had no severe consequences on cellular
physiology, including unaltered oxidative stress levels,
cellular growth, and viability in isolated cultures. However, the dis-
ruption of SCs caused massively reduced competitive cellular fit-
ness, which could be restored by the overexpression of Cytc. These
results thus demonstrate that the formation of SCs in living aerobic
cells increases the efficiency of cellular energy conversion, which
promotes an evolutionary advantage that can be selected for
(Berndtsson et al, 2020) (Fig 3C).
Results on increased electron transfer efficiency established by
this work align well with recent mathematical modeling of this
8 of 14 EMBO reports 24: e57092 | 2023
Andreas Kohler et al
process (Stuchebrukhov et al, 2020). Interestingly, further work
performed with yeast mitochondria in combination with cryo-EM
suggests that this kinetic advantage might be conferred by 2D diffu-
sion of Cytc between CIII2 and CIV in SCs rather than 3D diffusion
between separated, individual complexes (Moe et al, 2021) (Fig 3B).
In line with a previous proposal (Perez-Mejıas et al, 2019), it was
suggested that CIII2 + CIV SC formation provides a negatively
charged surface patch on top of this SC, on which the positively
charged Cytc can “roll”. These structural properties can also be
found in the mammalian version of this complex (Vercellino &
Sazanov, 2021). Hence, this mechanisms might present an alterna-
tive to substrate channeling, for which diffusion of Cytc within the
SC is favored by electrostatic interactions, but still allows a free
exchange with the universal soluble Cytc pool (Moe et al, 2021;
Vercellino & Sazanov, 2021, 2022; Fig 3B). However, the signifi-
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cance of an electrostatic interaction for 2D diffusion restricted to SCs
depends on the ionic strength of the local environment and the lipid
composition of the IMM. Currently, these aspects have not been
firmly determined, but are essential to establish the relevance of
such a 2D diffusion. Moreover, whether physiological or environ-
mental factors could modulate the ratio of Cytc 2D versus 3D diffu-
sion has not been investigated. In addition, limiting the 3D diffusion
of Cytc impairs mitochondrial function and cellular viability (Toth
et al, 2020). Of note, these results were obtained by reducing the
mobility of the whole Cytc pool and not only a potential subpopula-
tion interacting with SCs. In sum, current studies demonstrate that
the formation of CIII2 + CIV SCs increases the efficiency of electron
transport via Cytc. However, the exact modalities and biophysical
properties establishing this advantage remain to be investigated.
Mitochondrial respiratory complex stability and assembly
The structural interdependence between MRC complexes was first
observed in cells from patients with mitochondrial diseases, where
mutations affecting CIII or CIV frequently lead to a combined CI
deficiency (Lamantea et al, 2002; Fernandez-Vizarra et al, 2007),
suggesting that SCs may play roles in CI stability. The observation
that, in a CIII mutant, CI levels could be restored by treatment with
antioxidants, while SC-associated CI is not affected by high ROS
levels (Diaz et al, 2012), suggests that only free CI is highly sensitive
to ROS-induced damage and degradation.
Another hypothesis on the physiological significance of SCs
suggested that these large assemblies are scaffolds for the late stages
of CI biogenesis involving the incorporation of the NADH module to
the CI peripheral arm (Moreno-Lastres et al, 2012). However, this
hypothesis has been challenged by kinetic studies of CI assembly,
where a small amount of fully assembled CI could be detected
before the formation of SCs (Guerrero-Castillo et al, 2017). More
recently, it was shown that the lack of CIII prevents the incorpora-
tion of CI NADH module from completing the functional enzyme
(Protasoni et al, 2020). Moreover, the association of the late assem-
bly CI precursor with CIII and/or CIV was detected in mutant cell
lines and animal models (Alston et al, 2018; Adjobo-Hermans
et al, 2020; Fang et al, 2021). Additionally, data supporting the inter-
action of CIII and CIV subunits and assembly modules directly in
the SCs have been reported (Lazarou et al, 2009; Lobo-Jarne
et al, 2020; Tim on-Gomez et al, 2020). Taken together, increasing
evidence supports the existence of distinct but interconnected
assembly pathways for free complexes and SCs, a model referred to
 2023 The Authors
Andreas Kohler et al
as the cooperative assembly model (Fernandez-Vizarra & Ugalde,
2022).
Mitochondrial respiratory chain organization and
metabolic adaptation
The plasticity model of mammalian MRC organization proposes that
individual complexes assemble into SCs in a dynamic manner to
adapt to changing cellular metabolic needs (Acın-Perez et al, 2008).
However, despite the variety of MRC complexes distribution in
free form and SCs observed in different cell types and tissues
(Lapuente-Brun et al, 2013), the data reflect steady-state levels and
the dynamic nature of the MRC organization has not been experi-
mentally demonstrated. Alternatively, adaptation to the cellular
energetic requirements could be provided by the existence of inter-
connected assembly pathways, where incorporation of late assem-
bly catalytic modules into individual complex or SC pre-assembled
intermediates could modulate MRC organization (Fernandez-Vizarra
& Ugalde, 2022). Although the mechanisms behind these models are
yet to be fully disclosed, both scenarios suggest the involvement of
SC assembly/disassembly factors acting as sensors or assembly
checkpoints. As mentioned above, the only dedicated SC
assembly factor identified to date is SCAF1 (Cogliati et al, 2016).
There is a general agreement that in all cell lines and model organ-
isms, the absence of or mutations in SCAF1 lead to the lack of SC
CIII2 + CIV formation, while allowing the assembly of respirasomes
(Cogliati et al, 2016; Perez-Perez et al, 2016; Lobo-Jarne &
Ugalde, 2018). It is also established that the stability of respirasomes
and, particularly, larger SC structures is compromised to different
extents depending on the cell type and tissue (Cogliati et al, 2016;
Perez-Perez et al, 2016; Williams et al, 2016; Lobo-Jarne &
Ugalde, 2018). On the contrary, the question of whether the changes
in SC organization promoted by the absence of SCAF1 ultimately
lead to a metabolic adaptation remains to be fully answered. Some
studies concluded that SCAF1-dependent MRC remodeling is not
essential, neither to maintain mitochondrial bioenergetics nor to
cope with acute cellular stresses (hypoxia or oxidative stress) in
HEK293T cells (Perez-Perez et al, 2016; Lobo-Jarne & Ugalde, 2018).
Other studies showed that in U2OS cells, ER stress and glucose
deprivation stimulate mitochondrial bioenergetics and SC forma-
tion through protein kinase R-like ER kinase (PERK) by enhancing
SCAF1 levels through the PERK-eIF2a-ATF4 axis (Balsa
et al, 2019). Likewise, increased SC levels were associated with
mitochondrial efficiency and growth in severely hypoxic pancreatic
cancer (Hollinshead et al, 2020). An inconsistent picture was also
obtained from the study of animal models. These studies have
shown that SCAF1 promotes SC formation as well as metabolic
efficiency in zebrafish (Garcıa-Poyatos et al, 2020) and in some
mouse models (Lapuente-Brun et al, 2013; Shiba et al, 2017) but
not others (Mourier et al, 2014). One of these studies showed that
SCAF1 KO mice exhibit lower blood glucose levels after insulin or
pyruvate injection, which suggested that the protein could be
involved in the regulation of glucose homeostasis (Shiba
et al, 2017). However, whether the physiological manifestations
correlated with or were the cause of altered SC formation was not
fully defined. Work in zebrafish models showed that SCAF1-
deficient animals were smaller in size and had abnormal fat depo-
sition, phenotypes that were rescued by doubling the food supply
(Garcıa-Poyatos et al, 2020). This phenotype rescue correlated with
 2023 The Authors
EMBO reports
improved bioenergetics; however, possible SC-independent roles
of SCAF1 in fat metabolism in fish were not fully discarded.
Two more recent studies have added support to the concept of
MRC organization-dependent metabolic remodeling. One study
showed that two separate MRC organizations co-exist in human
cells and post-mitotic tissues, which the authors called C-MRC and
S-MRC. These are defined by the preferential expression of the CIV
COX7A isoforms, COX7A2 and SCAF1 (Fernandez-Vizarra et al,
2022), respectively. The SCAF1-dependent S-MRC organization is
characterized by the presence of the SCAF1-containing respirasome,
which accounts for approximately 50% of total CIII and CIV levels.
At the same time, the remaining CIII and CIV are equally distributed
between the CIII2 + CIV SC and free complexes. On the contrary,
the COX7A2-dependent C-MRC organization displays a relatively
low amount of the COX7A2-containing respirasome, no CIII2 + CIV
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SC, and abundant free CIII (~ 60% of total CIII) and CIV (~ 80% of
total CIV). The exclusive presence of one configuration or the other
in knock-out cells of the corresponding isoform led to some changes
in mitochondrial bioenergetics. However, no differences in respira-
tory parameters were observed between SCAF1 or COX7A2 knock-
out and wild-type cells, where the two MRC organizations co-exist.
The increment in respiratory rate supported by the addition of succi-
nate (a CII substrate) to permeabilized cells already respiring CI sub-
strates was shown to be lower in cells expressing the S-MRC
compared to C-MRC, suggesting that the electrons from CI saturate
the downstream MRC segment (Q to CIV) at a greater extent in the S
configuration. Preferential utilization of the NADH versus FADH2
route parallels with the fraction of total CIII and CIV distributed in
respirasomes. This suggests the existence of a kinetic advantage for
electron transfer within SCs possibly linked to the proximity of elec-
tron carrier binding sites. However, NADH- and FADH2-linked respi-
ration are not fully additive in either configuration, arguing against
Q pool partitioning. Moreover, the lack of COX7A2 causes a
decrease in total CIV amount and enzymatic activity by 50%, which,
although it does not affect endogenous and coupled respiration,
reduces spare respiratory capacity. This relevant confounding vari-
able can contribute to the observed decrease in CII minus CI- and
ATP-driven respiration in S-MRC cells. Lastly, the authors showed
that stimulation of pyruvate dehydrogenase (PDH) activity to pro-
mote a switch toward oxidative metabolism in human fibroblasts
reduces SCAF1 steady-state levels and favors the C-MRC configura-
tion. This observation establishes a direct link between the MRC
organization and cellular metabolism, controlled by PDH activity.
Intriguingly, while the study proposes that a metabolic shift toward
oxidative metabolism destabilizes SCAF1, data from human and
mouse tissues indicate that SCAF1 expression is induced by PGC1a
and enhances respiratory capacity (Benegiamo et al, 2022).
A study in humans has shown significant genetic variation in
SCAF1 in several tissues, most prominently in skeletal muscle
(Benegiamo et al, 2022). The most significant variant was an inser-
tion in the 30 untranslated region of the gene that creates a short-
repeated sequence and enhances SCAF1 expression. Human
myotubes carrying this genetic variant have enhanced SC levels and
respiratory capacity. One would expect that the S-MRC would accu-
mulate in these myotubes, but this was not determined. Neverthe-
less, an important finding was that this variant is associated with
lower body fat and improved cardiorespiratory fitness in humans
(Benegiamo et al, 2022). This observation was reproduced in a
EMBO reports 24: e57092 | 2023 9 of 14
EMBO reports
mouse model, where it was also shown that SCAF1 and SCAF1-
containing SCs are induced upon exercise, specifically in the skeletal
muscle, in a manner independent of PGC1a (Benegiamo et al,
2022). Taken together, these studies support the conclusion that
SCAF1 and SC levels correlate with metabolic fitness (Fig 3C),
although the cellular context may modulate the nuances of this
correlation.
Other proposed functions of respiratory chain supercomplexes
Most studies on SCs mention that the limitation of oxidative stress
could be a function of these assemblies. However, only very limited
evidence for such a role was provided. CI and CIII are the main pro-
duction sites of reactive oxygen species in mitochondria (Mur-
phy, 2009), and hence, it was postulated that the formation of the
respirasome somehow alleviates oxidative stress from these sites.
Indeed, an in vitro study suggests that the dissociation of CI + CIII2
SCs increases oxidative stress (Maranzana et al, 2013). In line with
these results, it was observed that the higher abundance of free CI
in astrocytes compared to neurons correlates with higher oxidative
stress found in astrocytes (Lopez-Fabuel et al, 2016). Unfortunately,
a molecular mechanism for such a potential reduction of reactive
oxygen species by SC formation has not been demonstrated, and
hence, this hypothesis is only based on correlations. A causative
role of SC formation to limit oxidative stress remains to be
determined.
Finally, it was also proposed that SCs prevent the aggregation of
proteins in the IMM (Blaza et al, 2014). Although this is feasible, as
the IMM presents one of the most protein-dense regions of a cell,
direct experimental evidence for this hypothesis is missing.
Summary
Since the discovery of SCs more than 20 years ago (Scha €gger &
Pfeiffer, 2000), significant advances in our understanding of these
gigantic assemblies have been made. In particular, the latest work
on the structural characterization of SCs presented a giant leap in
the field (Gu et al, 2016; Letts et al, 2016; Hartley et al, 2019;
Rathore et al, 2019; Vercellino & Sazanov, 2021; Zhou et al, 2022;
Klusch et al, 2023; Maldonado et al, 2023). Still, the physiological
functions of SCs remain a matter of debate (Milenkovic et al, 2017).
Recent reports have indicated that SC formation primarily enhances
the efficiency of electron transport. The emerging picture favors a
model in which SC formation, as such, does not modify the maximal
activity of individual complexes (Mourier et al, 2014; Berndtsson
et al, 2020), but optimizes their cooperation in the context of the
overall pathway.
Supercomplex formation constitutes another example for a
growing theme of gathering functionally related activities in com-
mon complexes or into proximal localization. This strategy, which
is widely employed in life, is used extensively in mitochondria
(Linden et al, 2020; Schulte et al, 2023) as exemplified by a super-
complex collecting enzymes mediating Q biosynthesis (Marbois
et al, 2009), the establishment of membrane subdomains where
early steps of MRC biogenesis are organized (Singh et al, 2020)
and mitochondrial RNA granules (Antonicka et al, 2013; Jourdain
et al, 2013), executing early steps of mtRNA maturation. However,
while all these organizations might not be essential for these
10 of 14 EMBO reports 24: e57092 | 2023
Andreas Kohler et al
In need of answers
• To fully unravel the functional interplay of respiratory chain com-
plexes in SCs and its mechanistic benefit, it might be necessary to
reconstitute purified SCs into proteoliposomes to study their perfor-
mance in a minimal system, free from confounding secondary
variables.
• The functional connectivity of the respiratory chain by mobile elec-
tron carriers is likely a key aspect of SC/respirasome formation. It
would be important to determine the absolute and relative concen-
trations of Cytc or Q, and RCs in order to test how they might deter-
mine the dependency on SC/respirasome formation to achieve
optimal electron transport rates.
• While this manuscript was under editorial proof, an article was
published where a more than 50% reduction in SC and respirasome
formation was achieved in a mouse model, without overly perturbing
respiration or physiology (Milenkovic et al, 2023). Hence, it would be
desirable for future studies to generate mutants in higher organisms,
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where the formation of SCs and respirasomes is completely abolished.
• Once these mutants are available, it would be very interesting to
score how they impact stress tolerance and competitive fitness.
• The interdependency of assembly of the different respiratory chain
complexes has suggested that mature complexes might facilitate
assembly of the partner complex through direct contacts. However,
assembly also occurs in the absence of the partner complex. To
unravel the contribution for the co-assembly pathways for overall
assembly, efficiency will be an important aspect of future research.
pathways to occur, they likely improve the overall biochemical
efficiency, possibly only with a small, but nevertheless selectable,
margin. While these small beneficial margins may be neglectable
or even sometimes undetectable in experimental setups and
under laboratory conditions, they have impacted selection during
evolution and seem to affect performance in mammalian tissues.
Particularly in the context of evolutionary pressure via
competition in natural environments and in stress scenarios (e.g.,
high temperature, hypoxic conditions, limited nutrients), these
subtle bioenergetic advantages of SC formation can be the basis
for selection. Employing the recent structural insights into respira-
tory SCs should allow engineering of further sets of genetic
mutants to fully unravel the intricacies and physiological impact of
SC formation.
Acknowledgements
We would like to thank the members of our laboratory for stimulating
discussions. This work was supported by Austrian Science Fund FWF (J4398-B
to AK), the Swedish research council (2014-4116 and 2018-03694 to MO), the
Knut and Alice Wallenberg foundation (2017.0091 and 2019.0319 to MO),
the National Institutes of Health R35 grant GM118141 (to AB), and the US
Army research office (W911NF-21-1-0359 to FF).
Author contributions
Andreas Kohler: Conceptualization; writing – original draft; writing – review
and editing. Antoni Barrientos: Conceptualization; writing – original
draft; writing – review and editing. Flavia Fontanesi: Conceptualization;
writing – original draft; writing – review and editing. Martin Ott:
Conceptualization; writing – original draft; writing – review and editing.
Disclosure and competing interests statement
The authors declare that they have no conflict of interest.
 2023 The Authors
Andreas Kohler et al
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