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BBA - Bioenergetics 1863 (2022) 148567
Available online 29 April 2022
0005-2728/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Cyanide resistant respiration and the alternative oxidase pathway: A
journey from plants to mammals
Riyad El-Khoury
a
, Malgorzata Rak
b
, Paule B´
enit
b
, Howard T. Jacobs
c
,
d
,
*
, Pierre Rustin
b
,
**
a
American University of Beirut Medical Center, Pathology and Laboratory Medicine Department, Cairo Street, Hamra, Beirut, Lebanon
b
Universit´
e Paris Cit´
e, Inserm, Maladies neurod´
eveloppementales et neurovasculaires, F-75019 Paris, France
c
Faculty of Medicine and Health Technology, FI-33014, Tampere University, Finland
d
Institute of Biotechnology, University of Helsinki, Helsinki, Finland
ARTICLE INFO
Keywords:
Plants
Mitochondria
Thermogenicity
AOX
Cyanide resistant respiration
Mammals
ABSTRACT
In a large number of organisms covering all phyla, the mitochondrial respiratory chain harbors, in addition to the
conventional elements, auxiliary proteins that confer adaptive metabolic plasticity. The alternative oxidase
(AOX) represents one of the most studied auxiliary proteins, initially identied in plants. In contrast to the
standard respiratory chain, the AOX mediates a thermogenic cyanide-resistant respiration; a phenomenon that
has been of great interest for over 2 centuries in that energy is not conserved when electrons ow through it.
Here we summarize centuries of studies starting from the early observations of thermogenicity in plants and the
identication of cyanide resistant respiration, to the fascinating discovery of the AOX and its current applications
in animals under normal and pathological conditions.
1. Introduction
Mitochondria are multifunctional organelles that play a key role in
many essential cellular processes, as well as being major sites of heat
production. They are a main source of the high-energy phosphate
molecule, adenosine triphosphate (ATP) that is synthesized by the pro-
cess of oxidative phosphorylation (OXPHOS), wherein substrate oxida-
tion via mitochondrial respiratory chain (RC) is coupled to ADP
phosphorylation [1]. In addition to the canonical elements of the RC,
mitochondria of many different organisms from all kingdoms contain
“auxiliary” proteins that participate in electron transport under specic
conditions (Fig. 1). Alternative oxidases (AOXs) and alternative NAD(P)
H dehydrogenases (NDX) represent two classes of these RC “auxiliary”
proteins that branch electron ow at various quinone reduction or
oxidation sites. AOXs complete electron transfer from quinones to oxy-
gen, bypassing RC complexes III and IV, while NDXs oxidize NADH/
NADPH, bypassing RC complex I or even providing a substitute for
complex I, whenever it is absent. [2,3]. Thus, the activity of auxiliary
proteins has a signicant impact on the tightness of coupling between
carbon catabolism and ATP synthesis. In various circumstances, they
confer signicant exibility to the RC, enabling organisms endowed
with such proteins to survive uctuating environmental conditions.
In this review, we summarize over a century of research conducted
on the cyanide-insensitive respiration mediated by the terminal oxidase
AOX. We will start from the initial observations of heat production in
plants. We will then describe the discovery of cyanide resistant respi-
ration and the formulation of the concept of parallel respiratory path-
ways, leading to the discovery of AOX itself. Finally, we will explore the
widespread occurrence of the enzyme beyond plants and review its
recent applications and future potential, notably in metazoans.
2. The road to the discovery of the alternative oxidase
2.1. Cyanide-resistant respiration
Thermogenicity in plants was rst described by Lamarck in 1778 in
the sterile part (spadix) of the inorescence of an arum lily, Arum itali-
cum [4] (Fig. 2). It was not until 1851 that a close association between
heat production and oxygen consumption was made by Garreau in the
same genus [5,6]. Although, it was long known that the oral spadix of
arum lilies has a remarkably high rate of respiration, the earliest pub-
lished measurements of respiration rates and of temperature therein
were provided in 1906 by Pfeffer [7]. Two years later, in 1908, Church
suggested that the excessive rise in temperature, amounting almost to
* Correspondence to: H. T Jacobs, Faculty of Medicine and Health Technology, FI-33014, Tampere University, Finland.
** Correspondence to: P. Rustin, Inserm UMR 1141, Bat Bingen, Hˆ
opital Robert Debr´
e, 48 Boulevard S´
erurier, 75019 Paris, France.
E-mail addresses: howard.jacobs@tuni. (H.T. Jacobs), pierre.rustin@inserm.fr (P. Rustin).
Contents lists available at ScienceDirect
BBA - Bioenergetics
journal homepage: www.elsevier.com/locate/bbabio
https://doi.org/10.1016/j.bbabio.2022.148567
Received 14 December 2021; Received in revised form 6 April 2022; Accepted 18 April 2022
BBA - Bioenergetics 1863 (2022) 148567
2
20 ◦C above ambient, might be vital for pollination by attracting polli-
nator insects to the owers [8,9].
Cyanide-resistant respiration was initially discovered not in plants,
but in protists, rst in Paramecium caudatum, by Lund in 1918 [10] then,
a year later, by Warburg in the unicellular green alga Chlorella sp. [11].
In 1929, concomitantly with the discovery of cytochromes, Genevois
described the rst respiration that was resistant to cyanide in plants, in
studies of sweet pea (Lathyrus odoratus) embryos (Fig. 2). The chemical
mechanism behind this respiration was, however, not addressed
[12–14]. Shortly thereafter, Okunuki described cyanide- and carbon
monoxide(CO)-resistant respiration in the pollen of Lilium auratum and
went on to formulate explicitly the concept of a dual respiratory
pathway, with a true alternative path for electron ow parallel to the
cytochrome pathway [15–17]. Okunuki did not, however, address the
issue of whether the proposed alternative pathway was coupled to ATP
production. The coupling of ATP formation to electron transfer in the
respiratory chain had been discovered around the same time by Engel-
hardt in avian erythrocytes [18]. In 1937, Van Herck reported the
marked resistance to cyanide of respiration by slices of the spadix of the
voodoo lily (Sauromatum), noting the high respiratory rates, but
ascribing the phenomenon to the existence of auto-oxidizable avo-
proteins [19,20].
In 1950, James and Beevers extended these discoveries to another
species of lily, Arum maculatum [9], showing that the respiratory system
in the spadix did not saturate even at 100% oxygen whereas that in the
peduncle saturated at 21% oxygen. They ascribed the observed differ-
ences to the existence of different oxidation mechanisms. They found no
evidence for the operation of a cytochrome system in the spadix, but
suggested that their ndings instead pointed to the existence of a low
oxygen-afnity, avin-dependent terminal oxidase.
2.2. Discovery of the alternative oxidase
James and Beevers demonstrated that cell-free extracts consumed
oxygen at high rates and reacted to different inhibitors similarly to the
intact tissues that they had studied [9]. Subsequently, Hackett and
Simon showed that particles prepared from spadix homogenates were
able to consume oxygen and to oxidize succinate as well as various Krebs
cycle intermediates [21]. In 1955, James and Elliott showed that the
particles isolated from Arum maculatum spadix were metabolically very
similar to mitochondria obtained from other plant and animal tissues
(Fig. 2) except that their respiration was resistant to cyanide [22]. In the
same year, Bendall and Hill showed that mitochondria from the Arum
spadix did contain the classical RC cytochromes, a, b and c. But in
addition, they identied a novel cytochrome component that did not
react with carbon monoxide, that they called b
7
, and proposed that it
could represent the alternate path of electrons to oxygen during cyanide-
resistant respiration [23]. Similar suggestions were put forward by in-
vestigators studying other plant species [17,24] but were challenged by
Chance and Hackett [25] who reported evidence for the partial reduc-
tion of cytochromes c and a in skunk cabbage mitochondria even in the
presence of high amounts of cyanide and azide, supporting the “excess
oxidase” hypothesis in place of a genuine alternate pathway [17].
However, Ikuma and colleagues soon obtained direct evidence for the
existence of two independent oxidases in mung bean hypocotyl mito-
chondria, distinguishable by their oxygen afnities [26]. Flux through
the alternative oxidase that they identied was characterized by a very
low apparent K
M
for O
2
(0.5
μ
M) [26]. They suggested that a ferro-
sulfoprotein with high oxygen afnity, rather than a terminal avo-
protein, was involved in the alternative pathway. Bendall and colleagues
then postulated that the alternate pathway in skunk cabbage mito-
chondria is a non-heme iron-containing protein of yet unknown struc-
ture [27].
Bendall and Bonner showed in 1971 that residual cytochrome oxi-
dase activity contributed only minimally to the large succinate-
dependent oxygen consumption rate observed after cyanide inhibition,
thus laying to rest the “excess oxidase” hypothesis. Furthermore, since
all three b-type cytochromes were almost completely reduced in
antimycin-inhibited mitochondria, the concept of a ‘b-shunt’ to oxygen
must also be abandoned. The addition of metal complexing agents
inhibited respiration in the presence of cyanide and antimycin, implying
that the alternate pathway involved an autooxidizable non-heme metal
complex [28]. These results were supported by kinetic experiments
conducted by Storey and Bahr in 1969 [29]. It was subsequently shown
that the cyanide-insensitive alternate pathway could be specically
inhibited by salicylhydroxamic acid (SHAM), and that it is signicantly
more active in state 4 (ADP-exhausted) substrate oxidation than in state
3 (in the presence of ADP) [30]. Although not a avoprotein as such, the
cyanide-resistant alternative oxidase did appear to be a metalloprotein.
Based on Mitchell’s 1975 hypothesis of the protonmotive ubiquinone
cycle [31], Rich and Moore proposed a similar scheme whereby the
alternate oxidase is in close association with the ubiquinone cycle in
Fig. 1. A schematic view of the proton-
motive and non-proton-motive respiratory
chain segments. The gure integrates the
alternative ux of electrons and the build of
proton-motive force, highlighting the rele-
vance or signicance of having respiration
coupled or uncoupled to proton-motive
force-generation. Note that the composi-
tion of each segment could be different ac-
cording to biological material, with a
number of components being present or
absent in specic cases (e.g. complex I being
absent in the yeast Saccharomyces cer-
evisiae). Action sites of inhibitors are indi-
cated in red. AOX, alternative oxidase; I-V,
the various complexes of the respiratory
chain; CN-, cyanide; ME, NAD+-dependent
malic enzyme; Nd
ex
, external NAD(P)H de-
hydrogenases; Nd
i
, internal rotenone-
resistant NADH dehydrogenase; SHAM,
salicyl hydroxamic acid; TCA, tricarboxylic
acid; UQ, ubiquinone. (For interpretation of
the references to colour in this gure
legend, the reader is referred to the web
version of this article).
R. El-Khoury et al.
BBA - Bioenergetics 1863 (2022) 148567
3
higher plants and that the alternate pathway oxidizes the QH’/QH2
couple whilst succinate dehydrogenase reduces it [32]. In 1978, Rich
succeeded in solubilizing and partially purifying the alternative oxidase
from A. maculatum. Moreover, it was stable in the solubilized state,
consistent with it being a protein, with its nal product being water
[33], not H
2
O
2
as initially thought [34]. Several other studies suggested,
however, that the involvement of lipoxygenase [35] or even fatty acid
peroxidation reactions could account for the observed cyanide-resistant
oxygen consumption in plants [36–38]. Such debates came to an end
when the enzyme was nally puried [39–42], and antibodies raised
against it [43,44] showed that homologues were widely distributed in
the plant kingdom and beyond [45–48].
Rhoads and McIntosh made a further major contribution by isolating
and characterizing the cDNA encoding the S. guttatum alternative oxi-
dase, which they designated aox1 [49] (Fig. 2). A homologous di‑iron
carboxylate protein, termed the plastoquinol terminal oxidase, was later
identied in Arabidopsis thaliana chloroplasts, indicating that the alter-
native oxidase is not conned to mitochondria [50,51].
2.3. Cyanide-insensitive respiration beyond the plant kingdom
In parallel to studies performed in plants, cyanide-insensitive respi-
ration was described in a number of other species prior to the actual
characterization of AOX. In 1950, Sanborn and Williams demonstrated
that the cytochrome pathway undergoes signicant quantitative and
qualitative adjustments during the lifespan of the giant silkworm, Hya-
plophora cecropia [52]. Specically, they showed that cytochromes b and
c are replaced by an unknown cytochrome component, which they
termed cytochrome x, in the larval stages, which disappears abruptly in
the prepupa [52]. In 1954, Pappenheimer and Williams showed that the
cytochrome x of the silkworm Platysamia cecropia was a b-type cyto-
chrome, subsequently referred to as cytochrome b
5
. A considerable
fraction of total electron ux linked to NADH oxidation was shown to
pass through cytochrome b
5
in this [53] and in a second silkworm spe-
cies, Bombyx mori even in the presence of cyanide or antimycin A [54].
In the body muscle of the parasitic nematode Ascaris suum, malate- and
succinate-driven respiration was also found to be insensitive to cyanide
and antimycin A [55–57]. However, cyanide-resistant oxygen con-
sumption in an organism does not necessarily imply the existence of an
alternative oxidase, as illustrated by the unique metabolic features of
A. suum [58]. As is now clear, AOX and the gene that encodes it is absent
from insects as well as from A. suum, though present in some other
nematodes [59].
Cyanide-insensitive respiration has also been noted in many protists.
In 1960, Grant and Sargent described a cyanide-resistant alternate
pathway represented by an L-
α
-glycerophosphate oxidase (GPO) system
that consumes oxygen independently from the classical cytochrome
pathway in the euglenozoan Trypanosoma rhodesiense [60,61]. The GPO
system was later shown to be linked to a plant-like alternative oxidase
[62]. In 1995, Chaudhuri et al. identied and partially puried the
trypanosome enzyme using antibodies raised against AOX from
S. guttatum [48]. Enzymatic activity was subsequently demonstrated in
E. coli for the putative AOX both from a trypanosome [63] and from an
apicomplexan [64]. AOX is now recognized as the exclusive terminal
oxidase responsible for oxygen-dependent respiration in the blood-
stream form of T. brucei [65,66] making it attractive as a potential drug
target [67–69].
AOX genes have been identied in many other eukaryotic protists,
such as in the green alga Chlamydomonas reinhardtii [70] and in jakobids
[2]. Whole genome analyses, taking advantage of high-throughput
sequencing combined with the development of powerful tools in bio-
informatics, have greatly extended the number of species that could
potentially harbor an AOX. Putative AOX genes may today be found
easily by BLAST searching in diverse stramenopiles, alveolates, haptista,
rhizaria, amoebozoa, other excavates and many other groups.
A major advance came with the discovery of the alternative oxidase
in bacteria (Fig. 2), initially in the Gram-negative bacterium Novos-
phingobium aromaticivorans in 2003 [71]. When expressed in E. coli the
recombinant enzyme was shown to confer cyanide-resistant terminal
oxidase activity [71]. Subsequent in silico exploration of metagenomic
data from ocean sampling expeditions indicated that the AOX gene is
widely distributed among bacteria [72–74], though has not been re-
ported in archaea. A functional, NO-resistant, terminal oxidase has
subsequently been identied in the marine bacterium Vibrio scheri
ES114 [75].
The discovery of the alternative oxidase in metazoans in 2004 [76]
represented another major breakthrough (Fig. 2). Today, AOX protein or
AOX-encoding genes have been found in over 150 metazoan species
distributed across many phyla, although are missing in vertebrates [3].
This widespread occurrence of AOX, including in the simplest multi-
cellular animals such as sponges, strongly favors its ancestral character
and implies that a gene loss event has taken place in vertebrates [3,59].
These observations highlight the issue of why AOX has been retained so
widely, yet not in vertebrates. Some clues are suggested from studies in
plants, whilst the impact of expressing invertebrate or fungal AOX in
model vertebrates has also been informative, as discussed below (Sec-
tion 4).
3. Structure, function and regulation of the alternative oxidase
The structural and functional biochemistry of AOX has been exten-
sively reviewed elsewhere, and the reader is referred to the relevant
Fig. 2. Timeline chart depicting some of the key discoveries from early observations of heat production and cyanide-resistant respiration in plants, to the discovery of
the AOX pathway and its recent application in mammals. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version
of this article).
R. El-Khoury et al.
BBA - Bioenergetics 1863 (2022) 148567
4
literature for details. Here we trace the history of studies of the enzyme
sourced from different taxa, and how it has impacted the most recent
work using AOX.
Early structure predictions, conrmed later by X-ray crystallog-
raphy, postulated that AOX is a non-heme diiron protein that belongs to
the diiron carboxylate superfamily [77–82]. The elucidation of the
crystal structures of the trypanosomal and plant (A. thaliana) enzymes
have provided structural insights into the nature of the active site, cat-
alytic mechanism and supramolecular organization of the enzyme
[83–85]. Both in plants and protists, the active site, buried in a hydro-
phobic cavity, is composed of the diiron centre with four glutamate and
two histidine residues. The Glu–X–X–His motifs stabilize the di‑iron
center and participate in catalysis [78,82–84,86,87], while other
conserved residues are essential for ubiquinol/ubiquinone and oxygen
binding [83–86,88]. Because of the recent interest in AOX as a potential
drug target, structural studies of the enzyme from different taxa will
undoubtedly intensify.
The supramolecular organization of AOX is more variable. Initial
Western blotting experiments using an AOX monoclonal antibody
detected multiple species [43,47,89], which were subsequently shown
to represent monomeric and dimeric forms of the enzyme, with dimers
linked by disulde bridges [79,90]. This organization appears to be
universal in plants, whereas fungal AOX exists as a monomer [91].
Following the discovery that AOX is widespread phylogenetically,
interest has turned to its biochemical and physiological functions. The
extensive literature on plant AOX provides a paradigm for understand-
ing its role(s) in other taxa, including metazoans [92] and for its use as a
tool to elucidate aspects of respiratory biology in species from which it
has been lost.
Like cytochrome oxidase, AOX catalyses a four-electron reduction of
oxygen to water, but does not compete with the canonical cytochrome
pathway, due to its very low afnity for its substrate, ubiquinol [93–95].
The partitioning of electron ux between the two pathways, in the
absence of inhibitors, is mainly governed by the reduction level of the
ubiquinone pools, which is inuenced by the available substrates and
the metabolic state of the cell. Oxygen concentration and the levels of
tricarboxylic acid (TCA) cycle intermediates are important regulators
(Fig. 3) [94–99]. This regulation enables signicant respiratory activity
to be maintained even in the presence of potent inhibitors of the cyto-
chrome pathway, such as cyanide, nitric oxide and sulphide [100,101].
The function of AOX in plant thermogenesis was the rst indication
of its existence, though this aspect has not been widely studied in other
taxa. However, it is an unavoidable consequence of its non-proton-
motive nature, and may have many other manifestations in biology.
Note that, even in organisms not endowed with AOX, up to 60% of the
energy released during respiration is converted to heat under normal
physiological conditions [102,103]. The thermogenic role of AOX is
likely to be a lively topic of future research, especially given the nding
that the operating temperature of mitochondria, at least in mammals, is
of the order of 50 ◦C [104].
In plants, AOX is essential for maintaining cellular homeostasis by
preserving mitochondrial activity in daylight, when ATP is efciently
produced by photosynthesis. AOX activation under such conditions
prevents mitochondrial RC blockage, which would otherwise inhibit all
metabolic reactions contingent on RC function, notably the TCA cycle
[105–107]. Similar considerations undoubtedly apply to photosynthetic
protists, but an abundance or over-abundance of ATP may occur also in
heterotrophic organisms where AOX could play a similar function. In
plants, this action of AOX limits the overproduction of reactive oxygen
species (ROS), which has been postulated as a more general role of the
enzyme in other taxa as well [108–111].
In plants, AOX also confers metabolic plasticity, enabling organisms
to adapt to various biotic and abiotic stress factors such as drought,
salinity, osmotic stress, nutrient deprivation, infection by pathogens or
extreme conditions of light and temperature: many of which have been
linked to ROS [112–126]. AOX also inuences fungal biology in ways
linked to ROS, in many cases with effects on pathogenicity [126–132].
How broadly AOX protects animals endowed with it against similar
stress conditions remains an open question.
Studies in plants and subsequently in other organisms have shown
that AOX expression and activity are usually under tight control [133],
which is easily understandable due to the potentially drastic effects of
the enzyme on cellular bioenergetics and heat generation. Regulatory
mechanisms so far elucidated can be divided into those that modulate
AOX gene expression and those that modify AOX enzyme activity post-
translationally. The number of nuclear genes encoding AOX varies, but
typically, as in Arabidopsis, the various AOX isoforms are functionally
distinct, differentially expressed in the tissues and life cycle, and respond
differently to environmental cues [92,134–138]. Similarly in fungi,
except where AOX is absent altogether [2,133], its expression can be
constitutive [133,139], variable according to life cycle stages [127,140],
or regulated by environmental factors and substrates [14,139,141–143].
Plant AOX is regulated chiey by organic acids [144], whereas both
fungal [141,144–147] and protistan [148,149] AOXs are modulated by
purine nucleotides, with a fungal-specic N-terminal tract of 20–25
amino acids suggested to confer this sensitivity [91]. Excellent reviews
dealing extensively with AOX regulation are available [92,126,133].
Fig. 3. Cyanide-resistance substrate oxidation by isolated mitochondria from
various plant species. The percentage of cyanide-resistant respiration was
measured under state 3 conditions (presence of ADP) using NADH (exogenous
source), succinate or malate (endogenous source of NADH). All substrates were
oxidized at roughly similar rates in the absence of cyanide.
R. El-Khoury et al.
BBA - Bioenergetics 1863 (2022) 148567
5
4. Heterologous expression of the AOX in animals: what have we
learned?
With the discovery of the AOX in metazoans in 2004, it became
possible to take up the challenge of expressing the enzyme in verte-
brates. Such an initiative sought to characterize the biochemical prop-
erties and physiological functions of metazoan AOX in different
contexts, using model organisms. The effects of AOX expression therein
might also shed light onto the conditions that could have led to the loss
of AOX in the course of evolution.
Shortly after the identication of the rst animal species harbouring
the AOX gene [76], Hakkaart and colleagues succeeded in expressing the
AOX of the tunicate Ciona intestinalis, a member of a sister group to the
vertebrates, in cultured human embryonic kidney (HEK-293T)-derived
cells [150]. The AOX protein was successfully targeted to mitochondria
and was engaged in respiratory electron ow only in the presence of
inhibitors that impose a high degree of reduction of ubiquinone. The
data suggested that metazoan AOX has similar biochemical properties to
the known plant enzymes, namely, low afnity for reduced ubiquinone
and possible post-translational regulation by organic acids [150]. AOXs
from a lamentous fungus and from plants were soon expressed suc-
cessfully in mammalian cells, as well [151–154].
C. intestinalis AOX was next expressed in pathological models. In
human cells harbouring specic mutations known to be responsible for a
number of cytochrome oxidase (COX)-related diseases, AOX expression
was able to alleviate various cellular consequences of COX deciency,
including impaired growth, compromised respiration and sensitivity to
oxidative stress [111,155,156]. Ubiquitous AOX expression in the fruit-
y Drosophila melanogaster produced no deleterious effects in wild-type
animals [157], but was able to counteract an array of deleterious con-
sequences at the whole-organism level. This included lethality triggered
by genetic manipulation of the cytochrome pathway [158] or by RC
inhibitors, such as cyanide and antimycin A [157], neurodegenerative
effects whether caused by genetic lesions affecting various COX subunits
[158] or induced by other mechanisms suggested to mirror aspects of
Alzheimer’s [159] or Parkinson’s diseases [157,160]. Decreased ROS
production was suggested as a mechanism, but AOX-expressing ies did
not show increased lifespan [161], contradicting at least the simplest
formulation of the ‘mitochondrial free-radical theory of ageing’. Unex-
pectedly, AOX expression also rescued cell migration defects during
development [162], but also had both negative and positive effects on
reproductive success under specic conditions [163–165], that remain
poorly understood.
AOX-expressing cells have also been used to explore important issues
in mitochondrial biochemistry. For example, expression of a fungal AOX
in a mouse cell-line bearing a mutation in the mt-CYTb gene, and thus
decient in complex III, established a link between ROS-induced com-
plex I degradation and the redox status of ubiquinone [166], enabling
RC organization to be remodelled according to the availability of
different substrates [166]. In contrast, ROS signaling was inferred not to
play a role in the stabilization of the transcription factor HIF1-
α
under
hypoxia in cultured HEK293-derived cells expressing the Ciona enzyme
[167]. AOX also promoted the migration of mammalian cultured cells
[162].
A further advance was achieved in 2013 by the rst successful
expression of AOX in a mammal [168]. As previously observed in
cultured human cells, the C. intestinalis AOX protein was successfully
targeted to mitochondria in different mouse tissues without competing
biochemically, under standard conditions, with the canonical RC, but
becoming functional upon blockade of the cytochrome segment. Mice
harbouring widely expressed AOX showed normal growth and devel-
opment, produced healthy offspring in normal numbers, and exhibited
increased resistance to otherwise lethal doses of gaseous cyanide [168].
However, since the mice were generated using a lentivector transduction
strategy, which resulted in the insertion of the AOX transgene at mul-
tiple genomic sites, long-term maintenance of the model was not
feasible. To overcome this problem, Szibor and colleagues generated a
genetically tractable mouse expressing a single copy of the C. instestinalis
AOX inserted into the non-essential Rosa26 locus [169], as well as a
conditionally activatable version thereof [170]. The AOX
Rosa26
mouse
showed stable, ubiquitous, and substantial level of expression of the
AOX. Moreover, the enzyme was functional and conferred resistance to
inhibitors of the cytochrome pathway.
Widespread expression of AOX in the AOX
Rosa26
mouse again did not
affect physiological processes under unstressed conditions [169].
However, when combined with various pathological models, AOX
expression produced a variety of outcomes, some of them unexpected,
providing insights into pathophysiological mechanisms. For example,
the use of the AOX
Rosa26
mouse demonstrated mitochondrial involve-
ment in lung damage caused by chronic exposure to toxic smoke [171],
as well as in a sepsis model [173]. Conversely, AOX was unable to rescue
a model of inammatory cardiomyopathy, and even exacerbated the
pathology [172].
In mouse models of RC disease a similarly mixed picture has
emerged. AOX prevented lethal mitochondrial cardiomyopathy in a
mouse model of GRACILE syndrome, caused by a mutation in an as-
sembly factor of RC complex III, Bcs1l [174], but worsened the pheno-
type of mouse models of COX-decient mitochondrial myopathy [175]
and of cardiac ischemia/reperfusion injury [176]. In the latter cases,
AOX abrogated ROS signaling, blocking repair and regeneration pro-
cesses, and providing a possible clue as to why AOX was lost from ver-
tebrates. ROS production from complex III was similarly implicated as
an essential trigger in hypoxia signaling in the lung, using AOX
Rosa26
mice in an ex vivo model of hypoxic pulmonary vasoconstriction [177].
AOX-expressing mice have also begun to be exploited to address
longstanding issues in mammalian mitochondrial bioenergetics. For
example, mice expressing a fungal AOX have been used to conrm that
the complex I +III supercomplex sequesters a portion of the mitochon-
drial ubiquinone, which modulates the oxidative capacity of complex I
[178]. Similar ndings have been made in mice expressing the Ciona
enzyme [179].
5. Conclusions and perspectives
The discovery of the AOX brought to an end a century of vigorous
debate on the exact nature of the machinery responsible for mitochon-
drial cyanide-resistant respiration, initially described in plants. The
development of powerful sequencing technologies combined with sig-
nicant progress in bioinformatics, ushered in a new era that led to the
realization that AOX is extremely widespread phylogenetically. X-ray
crystallography and functional enzymology then allowed its structure
and reaction mechanism to be elucidated. The discovery of AOX in
metazoans, but excluding vertebrates, has enabled heterologous
expression of the enzyme in mammals. Leveraging the unique metabolic
properties of the AOX has opened a new window on mitochondrial
biochemistry and enabled pathophysiological mechanisms associated
with RC dysfunction to be explored in a novel way. AOX may yet have
therapeutic potential [180], if the ways in which it interferes with cell
signaling and repair can be fully understood and mitigated.
In the light of the available and accumulating data in mammals, the
issue of the evolutionary loss of AOX can be divided into two rather
independent questions: rst, what are the potential detrimental effects
of AOX that are inextricably linked with vertebrate biology, and second,
if AOX was lost simply because the benets that it provides to organisms
such as plants and lower eukaryotes were no longer operative, what was
it about the vertebrate lifestyle that removed these constraints? Only a
better understanding of the biochemical and thermodynamic properties
of the AOX of lower metazoans and its physiological repercussions in
mammalian models will provide us with clear answers to these
questions.
Lastly, the history of research on AOX is a vivid illustration of the
importance of serendipity in science. Whilst genome analysis would
R. El-Khoury et al.
BBA - Bioenergetics 1863 (2022) 148567
6
have revealed the existence of the AOX gene in any case, without the
century of prior research on plant respiration and thermogenesis it
would have remained one of the thousands of orphan genes awaiting
proper characterization. Its use as a tool to understand mammalian
biochemistry and cellular functions, as well as human pathophysiology,
would likely be far in the future. Instead, research on the respiration of
plant tissue slices and on how organisms generate heat has led to
unpredicted insights into human biology with potential applications in
medicine.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
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