Physiol Rev 95: 219 –243, 2015
doi:10.1152/physrev.00006.2014
ELECTRONIC CONNECTION BETWEEN THE
QUINONE AND CYTOCHROME C REDOX POOLS
AND ITS ROLE IN REGULATION OF
MITOCHONDRIAL ELECTRON TRANSPORT AND
REDOX SIGNALING
Marcin Sarewicz and Artur Osyczka
Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian
University, Kraków, Poland
Sarewicz M, Osyczka A. Electronic Connection Between the Quinone and Cytochrome c
Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox
Signaling. Physiol Rev 95: 219 –243, 2015; doi:10.1152/physrev.00006.2014.—Mitochondrial respiration, an important bioenergetic process, relies on operation of four
membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc1 or ubiquinol:
cytochrome c oxidoreductase), provides an electronic connection between these two diffusible
redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with
one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme
implicate that in addition to its well-defined function of contributing to generation of proton-motive
force, cytochrome bc1 may be a physiologically important point of regulation of electron flow acting
as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic
conditions. These features include the following: the opposing redox reactions at quinone catalytic
sites located on the opposite sides of the membrane, the inter-monomer electronic connection that
functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system,
as well as the potential to generate superoxide and release it to the intermembrane space where
it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding
how cytochrome bc1 may accomplish this regulatory physiological function, what is known and
remains unknown about catalytic and side reactions within the quinone binding sites and electron
transfers through the cofactor chains connecting those sites with the substrate redox pools. We
also discuss the developed molecular mechanisms in the context of physiology of mitochondria.
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.
XI.
INTRODUCTION
PRINCIPLES OF ELECTRON TRANSFER...
QUINONES AND THE Q POOL
CYTOCHROME c AND THE C POOL
REDOX CONNECTION OF THE Q POOL...
COFACTOR CHAINS OF CYTOCHROME...
INTERACTIONS OF QUINONE AND...
GENERATION OF REACTIVE OXYGEN...
H-SHAPED ELECTRON TRANSFER...
COMPOUNDS THAT INFLUENCE THE...
CONCLUSIONS
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I. INTRODUCTION
Energy conversion is one of the fundamental processes supporting life of all organisms. It gives power to maintain homeostasis inside each cell and among cells. To achieve this, the
process of energy conversion itself must be efficient, safe, and
appropriately regulated. As the universal source of energy directly available for the cells to do useful work is stored in the
concentrations of ATP, ADP, and Pi that are kept far from
their equilibrium concentrations, the bioenergetic processes
evolved to use the energy released from various chemical or
physical processes to power the synthesis of ATP from ADP
and Pi. While some amounts of ATP can be synthesized by
water-soluble enzymes, the major fraction of ATP synthesis is
associated with the operation of enzymes that are assembled to
form specific bioenergetic paths (194). These enzymes are embedded in biological membranes involved in energy conversion (bioenergetic membranes). The factor that unifies a diversity of substrates used by these enzymes and a diversity of
molecular mechanisms of conversion of these substrates is a
proton-motive force (PMF), a central element of Peter Mitchell’s chemiosmotic theory (176, 179). PMF depends on the
difference of pH values over a bioenergetic membrane (⌬pH)
and the membrane potential (⌬⌿; difference in electrical po-
0031-9333/15 Copyright © 2015 the American Physiological Society
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MARCIN SAREWICZ AND ARTUR OSYCZKA
tentials between two aqueous compartments separated by a
membrane). PMF, generated by the enzymes that use external
source of energy to transfer protons between compartments
separated by a bioenergetic membrane, forces ATP-synthase
to convert ADP and Pi into ATP.
Generation of PMF commonly relies on operation of several enzymes that are functionally linked together. In mitochondria,
such assembly is located in the mitochondrial inner membrane
forming the mitochondrial respiratory chain (FIGURE 1). The
chain usually consists of four complexes, termed complex I to IV,
which catalyze the transfer of electrons from substrates, such as
NADH or succinate, to the final electron acceptor O2. Three of
these complexes (complex I, III, and IV) couple the electron transfer with proton translocation across the membrane.
These enzymes catalyze step-by-step electron transfer from the
molecules of lower redox midpoint potential1 to the molecules
of higher midpoint redox potential, and the stepwise release of
energy in these reactions is used to translocate protons across
the membrane. Within the protein, electrons travel through
the redox cofactors (like hemes or iron-sulfur clusters) that
form chains connecting various catalytic sites (4, 183). The
1
The term redox midpoint potential (Em) of a redox couple commonly
used in biology is a derivative of “standard redox potential” (E0) and
refers to the “actual redox potential” (Eh) at pH other than 0 (typically pH
7) for which concentration of oxidized and reduced forms are equal. E0
is equal to Eh at pH 0, under which concentrations of oxidized and
reduced forms are equal and their activities are maintained at unity.
cofactor chains and catalytic sites of respiratory complexes are
linked together by small electron carriers that move between
complexes. These carriers form two redox pools, each confined to the environment of different polarity. One pool,
formed by quinone molecules (the Q pool), is confined to the
lipid phase of inner mitochondrial membrane while the second
pool, formed by cytochrome c (the C pool), is confined to the
water phase of mitochondrial intermembrane space. Such a separation prevents energy-wasting direct electron exchange between the pools despite a significant difference in redox midpoint
potential of quinone and cytochrome c (FIGURE 2). This is critically important for the efficiency of mitochondrial respiration.
It is equally important that electrons do exchange between these
two pools in the energy-conserving process. Such connection is
secured by the operation of one of the complexes of mitochondrial respiratory chain. This complex, named cytochrome bc1
(EC 1.10.2.2; quinol-cytochrome c-reductase, complex III), catalyzes a net reaction of oxidation of ubihydroquinone (UQH2)2
2
Abbreviations for quinone nomenclature used in the text: USQ⫺,
semiquinone anion; USQH, protonated semiquinone; USQ, general description of semiquinone radical regardless of its protonation state (in
some cases it is used when protonation state is unknown); USQo,
semiquinone in the Qo site; USQi, semiquinone in the Qi site; UQ,
ubiquinone (fully deprotonated); UQH2, ubihydroquinone (ubiquinol); Q
or quinone, general reference to quinone irrespective of the redox and
protonation state of the molecule.
FIGURE 1. Components of mitochondrial electron transport chain: membranous complexes I–IV linked by
diffusible quinone (yellow sticks) in the membrane (forming the Q pool) and cytochrome c in the intermembrane
space (forming the C pool). Cytochrome bc1 (complex III) connects electronically the Q pool with the C pool.
220
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REGULATION AND FUNCTION OF CYTOCHROME bc1 IN MITOCHONDRIA
400
cytochrome c2
cytochrome c
Midpoint redox potential [mV]
300
200
100
ΔG
qu
ino
ne
FIGURE 2. Dependence of average Em of the Q pool (green)
and Em of mitochondrial C pool (red) on pH. The energy associated with cytochrome bc1-catalyzed electron transfer between the pools (⌬G) increases upon rising pH. Bacterial cytochromes c2 (blue) have generally higher Em than mitochondrial counterparts.
0
-100
-200
-300
5
6
7
8
9
10
11
pH
and reduction of cytochrome c (20, 56, 65). The energy
released during this electron transfer powers the reaction of
oxidation of UQH2 and reduction of ubiquinone (UQ),
each taking place at the distinct catalytic site located on the
opposite side of the membrane. At one site (Qo site) UQH2
is oxidized and protons are released, while at the other site
(Qi site) UQ is reduced and protons are uptaken. The joint
action of these two catalytic sites, as originally proposed by
Peter Mitchell in his Q cycle (177), leads to a net translocation of protons across the membrane contributing to PMF.
This defines a basic bioenergetic role of this enzyme.
An unusual feature of this enzyme, described on the basis of
the Q cycle, is that the product of one catalytic site becomes
the substrate for the second site, and vice versa (i.e., UQ
produced in the Qo site is the substrate for reaction in the Qi
site, while UQH2 produced in the Qi site is the substrate for
reaction in the Qo site). This, together with the fact that
these sites are connected with the Q and C pools, makes
cytochrome bc1 a potential point of the regulation of the
mitochondrial respiration at molecular level.
The other potential role of this enzyme is related to the
observation that under some conditions cytochrome bc1
can generate reactive oxygen species (ROS) in the form of
superoxide (26, 83). In fact, cytochrome bc1 is considered a
second, after complex I, source of ROS delivered from components of mitochondrial respiratory chain (33, 47). ROS,
according to current views, cannot only be damaging to the
cells, but also, in certain instances, were postulated to be
important in cellular signaling (97, 122, 191, 240). Indeed,
the superoxide generated by cytochrome bc1 can be related
to both types of effects associated with ROS generation.
Understanding of the energetic and regulatory roles of cytochrome bc1 requires the unification of knowledge on how
physiological and energy-conserving reactions are supported while the energy-wasting and often thermodynamically favorable side reactions are avoided. The following
text discusses current view on efficiency, safety, and regulation of the physiological connection between the Q pool
and the C pool secured by cytochrome bc1 in the context of
mitochondrial function. It begins with a brief outline of
basic principles of electron transfer and the organization
of electron transfer chains followed by a summary of selected properties of quinone and cytochrome c. It then discusses the molecular and physiological aspects of the mechanisms of cytochrome bc1 interactions with its substrates,
of quinone redox reactions within the catalytic sites, and of
ROS production by cytochrome bc1 to end with an emerging new physiological perspective afforded by recent studies
that exposed a dimeric function of the enzyme.
II. PRINCIPLES OF ELECTRON TRANSFER
BETWEEN COFACTORS IN PROTEINS
It is generally accepted that biological electron transfer is a
process of tunneling, the quantum mechanical phenomenon
in which an electron can cross a barrier of insulating protein
medium between the redox active donor and acceptor. The
rate of the tunneling is the product of two terms. The first is
an electronic term arising from the strength of the coupling
of electron donor/acceptor wave functions that decreases
exponentially upon increasing the distance between donor
and acceptor. The second term depends on both the driving
force (⌬G, proportional to the difference in redox midpoint
potentials between donor and acceptor) and the reorgani-
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MARCIN SAREWICZ AND ARTUR OSYCZKA
zation energy required to change the nuclear coordinates
upon electron transfer (14, 170, 184, 185, 203).
Within the protein matrix the dominant factor that influences the electron transfer rate is the distance between redox centers. The physiologically relevant electron transfer
rate (usually millisecond or less to support the turnover of
the enzymes) requires that the edge-to-edge distance between donor and acceptor should not exceed ⬃14 Å (203).
Indeed, the geometric arrangement of cofactors that form
chains in known proteins follows this rule, and the interacting redox centers are usually within 14 Å (182, 183). The
reactions that are unproductive and adverse from physiological point of view usually face distances larger than 14 Å,
which makes the rates slow enough to be insignificant.
However, there are cases where such long-distance suppression of unproductive reactions is not possible and other
mechanisms must be involved. One of the intriguing examples includes the Qo site of cytochrome bc1 (199, 200).
The second important factor influencing electron transfer
rate is the driving force, which in general should be considered as the free energy released during a spontaneous reaction of electron transfer from the low- to high-potential
cofactor. Thus, to push electron along the chain in specific
direction, the consecutive cofactors exhibit the increasing
midpoint redox potentials. Interestingly, many cofactor
chains possess energetically unfavorable “uphill” steps related to the presence of cofactors of lower potentials flanked
by the two cofactors of higher potentials. Although the
physiological significance of such arrangement is not clear,
it appears that strikingly uphill electron transfer can occur
at functionally relevant times provided that there is an adequate proximity between the centers and an overall driving
force along the chain (5, 158, 203).
and this observation was adopted by Peter Michell to propose the mechanism of protonmotive Q cycle (177, 178).
Nowadays, apart from the bioenergetic function, quinones
are considered to be engaged in a variety of cellular functions that are not only associated with their redox properties but also with the capability to change the fluidity of the
lipid bilayers (48, 96, 256). The later point is supported by
the fact that occurrence of quinones is not restricted to
mitochondrial membrane (91), and the changes in their
content is also correlated with age and with several diseases
(10, 86, 87, 138, 156, 167, 263).
While eukaryotic cells use two major types of quinones as
elements of energy conversion systems: ubiquinones (or coenzyme Q) in respiration and plastoquinone (present in
membranes of chloroplasts) in photosynthesis, prokaryotic
cells can also use other types of quinones for those purposes. One example includes a variety of menaquinonerelated compounds (198) that are evolutionary more ancient than ubiquinones and plastoquinones and, by virtue of
their low redox midpoint potential, are proposed to be engaged in bioenergetics of cells living in reducing atmosphere
of preoxygenic eon (227). On the other hand, in humans,
some menaquinones (identified as vitamin K) are also present but play other roles (24, 28).
A. Redox Properties
Benzoquinone ring of quinone may exist in nine different
redox states (212) that result from three possible electronic
and three protonation states (FIGURE 3). However, only six
UQ
USQ-
UQ2-
UQH+
USQH
UQH-
UQH22+
USQH2+
UQH2
III. QUINONES AND THE Q POOL
Quinone derivatives are ubiquitous constituents of almost
all lipid membranes of all organisms. Only rare exceptions
are recognized in some prokaryotic cells of obligatory fermentative bacteria and methanogenic archae (198). Quinones are redox active compounds with capability to uptake/release two protons and two electrons (213, 214). The
transitions between reduced and oxidized form involve formation of an intermediate semiquinone (USQ) which is an
unstable oxygen-centered free radical (161).
The role of isoprenoid quinones in the bioenergetics of cells
was first proposed in the late 1950s (58). The observation
that extraction of quinones from bioenergetic membranes
inhibited the activity of mitochondrial complexes led to the
conclusion that quinones are responsible for shuttling electrons between respiratory complexes (92, 146, 196). Further experiments revealed that semiquinone intermediate is
formed during the catalytic cycle of cytochrome bc1 (265),
222
Em(UQ/UQH2) = [Em(USQ/UQH2) + Em(UQ/USQ)]/2
Kstab(USQ) =
[USQ]2
[UQ] [UQH2]
FIGURE 3. Possible redox and protonation states of quinone molecules and definition of Em of UQ/UQH2 couple and Kstab of semiquinone. The states that can be detected experimentally in solution or
in catalytic sites of proteins are shown in black. Gray represents the
states not present in biological environment. Vertical and horizontal
arrows denote steps of protonation and reduction of the molecules,
respectively. See References 115, 212.
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REGULATION AND FUNCTION OF CYTOCHROME bc1 IN MITOCHONDRIA
states were detected experimentally. Among these six states,
only two are found to be relatively stable in bulk lipid phase
(115, 281). However, in catalytic sites, the number of stable
states can be larger. The redox midpoint potential of
ubiquinone/ubiquinol (UQ/UQH2) couple [Em(UQ/UQH2) in
FIGURE 3] is usually described as an average redox potential
of two redox couples: ubiquinone/semiquinone (UQ/USQ)
and semiquinone/ubihydroquinone (USQ/UQH2) (217).
The difference between the redox midpoint potentials between these two couples defines the stability constant for
USQ (Kstab in FIGURE 3): the larger the difference, the lower
equilibrium concentration of USQ (79, 200). Kstab of USQ
in bulk lipid phase is very low; therefore, in membranes
only, UQ and UQH2 forms are present, at least in physiological pH. Nevertheless, the quinone binding sites within
proteins often stabilize USQ as intermediate state of the
catalytic cycles of the enzymes (115, 161). The degree of the
stabilization of USQ may differ significantly from site to
site.
B. The Q Pool
The size of the Q pool (160) may vary depending on type of
membrane, organism, and tissue (1). In bioenergetic membrane, quinones are in excess in relation to the membranous
complexes of electron transport chain. In mitochondria of
mammals and plants, the estimated amount of quinone
molecules per cytochrome oxidase is in the range of 6 – 8,
while in yeast it is larger (⬃36) (124). In humans, the size of
the Q pool has been found to increase within the first 20
years of life, then it decreases to eventually reach the size
that is smaller compared with the size after birth (138). The
size of the Q pool may be modified by certain diseases and
even changed upon the progress of a disease (256). In general, these types of changes are expected to influence the
bioenergetic function of mitochondria and perhaps also
vary the contribution of the Q pool in the overall antioxidative activity of mitochondria.
One of the most important parameters of the Q pool is its
redox state defined as the ratio of UQ to total quinone
content in membrane. The redox state of the Q pool is
directly related to the bioenergetic state of the cells and is
considered as one of the markers of oxidative stress (246,
271). The redox state of the Q pool is expected to change
depending on the accessibility of the cells to oxygen, as for
example observed in hypoxia where level of UQ increases in
some tissues (105). Interestingly, hypoxia also induces a
decrease in a size of the Q pool. The redox state of the Q
pool may also change in diseases, as reported for cardiovascular diseases or cancer (256).
Early experiments on the kinetics of electron transfer revealed that the Q pool creates rather homogeneous ensemble of molecules rapidly diffusing within lipid bilayer, being
in equilibrium with binding sites of proteins (105, 146). An
alternative view assumes the compartmentalization of quinone molecules into separate pools within the membrane
(107). The estimation of the amount of quinone molecules
that are bound to the proteins in mammals is in the range of
10 –32% of the total quinone (106, 159). Mobile molecules
of quinones in membrane can collide with each other, which
theoretically can lead to self-exchange of electrons between
the colliding molecules. This reaction, however, in lipid
environment seems unlikely, or occurs at a very low rate, as
it requires deprotonation of UQH2 (213, 214).
C. Other Functions of Quinones
Quinones, besides playing a crucial role in energy conversion, are one of the most important antioxidants in the cells.
It has been shown that UQH2 is effective in inhibiting both
the initiation and propagation of lipid peroxidation chain
reaction (18). In these reactions, UQH2 undergoes one-electron oxidation to USQ by reducing perferryl radicals or
lipid peroxyl radicals. As USQ is a quite reactive electron
donor for molecular oxygen, it must be neutralized effectively. In humans, USQ is reduced to UQH2 by ␣-tocopherol (vitamin E). However, there are evidences that UQH2 is
still an efficient antioxidant compound even in the absence
of vitamin E (104). This requires the existence of other
mechanisms of neutralization of semiquinone radicals without engagement of vitamin E, possibly via dismutation (25).
The interest in antioxidant properties of quinones became
inspiration for synthesis of water-soluble derivatives such
as MitoQ or SkQ (7, 8, 134, 235). These compounds penetrate into mitochondrial intermembrane space where they
undergo reduction to hydroquinone form by the enzymes of
respiratory chain. At low concentrations, SkQ was shown
to prevent hydroxyl-radical-mediated cardiolipin peroxidation and inhibited ROS-induced apoptosis and necrosis of
human fibroblasts (7, 235).
It is now usually considered that the antioxidant properties
of quinones may have a beneficiary effect on health and the
lifespan. This has drawn the attention of the pharmacological industry to produce dietary supplements containing
ubiquinone (coenzyme Q). However, understanding of the
complex effect of quinone on an organism and its relation
to the lifespan is still incomplete, especially in the view of
reports of the increased lifespan of the nematode Caenorhabditis elegans under ubiquinone deficiency proposed to
affect metabolism of the nematode cells (155, 180).
Another possible function of quinone is related to the action
of the uncoupling proteins, which in the inner mitochondrial membrane translocate H⫹ down the electrochemical
proton gradient. This transport, not coupled to oxidative
phosphorylation, leads to the dissipation of energy stored in
PMF and generates heat. It was reported that quinone is an
obligatory cofactor for uncoupling protein function (85),
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MARCIN SAREWICZ AND ARTUR OSYCZKA
and the regulation by quinone depends on its redox state
(243). Indeed, UQH2 but not UQ functions as a negative
regulator of uncoupling proteins inhibition by purine nucleotides (267).
In addition to the functions discussed above, this molecule
appears to be engaged in other molecular reactions of the
cell, as for example in modulating the action of mitochondrial permeability pore (101). The detailed description of all
postulated functions of quinones is beyond the scope of this
review.
IV. CYTOCHROME c AND THE C POOL
Mitochondrial cytochrome c, since its description by Keilin
in 1925 (139), became one of the best-characterized proteins (181, 229). It is a small, water-soluble, globular protein containing one high-potential heme that undergoes one
electron oxidation/reduction. In mitochondrial respiration,
cytochrome c works as a mobile one-electron carrier connecting cytochrome bc1 with complex IV (119). In living
cells, the localization of cytochrome c is restricted to the
intermembrane space of mitochondria. Any release of this
protein to cytoplasm triggers an apoptotic pathway in
which cytochrome c becomes a part of apoptosome which
activates capsase-9 (3, 166, 211). Cytochrome c also appears to act as a cardiolipin oxygenase during early stages of
apoptosis (137).
Cytochrome c in solution can undergo an electron selfexchange reaction that refers to an electron transfer between cytochromes c without involvement of enzymes (81,
116, 170, 181). The physiological meaning of this reaction
is not clear. In the textbook schemes of mitochondrial electron transport chain, this reaction is usually not considered;
as for mitochondrial cytochrome c, it is relatively slow.
Consequently, the connection between cytochrome bc1 and
complex IV is described as a process in which a single molecule of cytochrome c takes electron from cytochrome bc1,
then this molecule diffuses to complex IV where it undergoes oxidation. However, taking into account the effect of
macromolecular crowding (89) and high concentration of
cytochrome c in the intermembrane space (0.1–5 mM)
(102), the self-exchange of electrons between cytochrome c
molecules in the pool might also take place. In this context,
the spatial distribution of electrons within the C pool, involving both the diffusion of molecules and the self-exchange reaction, would differ from the spatial distribution
of electrons within the Q pool involving just the diffusion of
quinone molecules that do not undergo self-exchange at
significant rates. Interestingly, the measured rates of selfexchange reaction between bacterial cytochromes c, which
have a lower net charge comparing to mitochondrial cytochrome c, is much faster than that measured for horse heart
cytochrome c (181).
224
The alternative view of the transfer of electron between cytochrome bc1 and complex IV evokes the idea of supercomplexes (27, 154), which when formed create different conditions of electronic connection. According to this concept, quinone and cytochrome c molecules are confined to form small
subpools operating just within boundaries of the supercomplexes. However, several studies, including recent time-resolved kinetics on intact cells, suggest that diffusion of cytochrome c is not restricted and the protein is not trapped
within supercomplexes (119, 252).
The relatively high redox potential of the heme c and the
positively charged surface near the heme crevice make cytochrome c an effective reactant for superoxide anion (41,
172). This reaction seems advantageous over the dismutation reaction as the superoxide anion is converted back to
oxygen instead of hydrogen peroxide. Thus high concentration of cytochrome c in the intermembrane space provides a
very efficient and safe way of neutralizing potentially deleterious superoxide that is released into the intermembrane
space by cytochrome bc1. For this reason, cytochrome c is
considered as an ideal antioxidant (102, 205).
V. REDOX CONNECTION OF THE Q POOL
WITH THE C POOL
To effectively drive the electron transport through the mitochondrial respiratory chain, the value of the midpoint
redox potential (Em) of the Q pool is set in the middle
between the Em values of the reducing equivalents that
come from the tricarboxylic acid cycle and the Em of C pool
that delivers electron for the terminal electron acceptor
(161, 183). Cytochrome bc1 as a point of connection between the Q and C pools uses energy stored in the difference
in redox potentials of quinone and cytochrome c and their
physical separation by confinements to chemically unmixable phases.
The Em of the Q pool is ⬃90 mV at pH 7 and, because
oxidation/reduction of quinone involves deprotonation/
protonation, decreases by ⬃60 mV when pH increases by 1
(245, 258). The Em of the C pool is ⬃260 mV at pH 7 (171)
and does not change significantly with pH. Because the Em
of the Q pool changes with pH while the Em of the C pool
does not, the energy associated with electron transfer (⌬G)
from the Q pool to the C pool should in principle increase as
pH rises (FIGURE 2). This could in part account for the
observed pH dependence of rate of cytochrome bc1 catalysis (35, 125, 199, 201).
Because the redox state of the Q pool and the C pool can
dynamically change in the cell, the real reducing power of
the Q pool and oxidizing power of the C pool depends not
only on Em but also on the actual ratio of oxidized to
reduced components of the pool that establishes the Eh of
the pool according to Nernst equation (194). For example,
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REGULATION AND FUNCTION OF CYTOCHROME bc1 IN MITOCHONDRIA
in healthy humans, the Q pool is generally above half-reduced, but in some tissues such as pancreas, it is almost fully
reduced which makes Eh much lower than Em (249).
The electron exchange between the Q pool and C pool
through cofactor chains of cytochrome bc1 will depend on
the actual conditions that encompass the redox states of the
pools and the membrane potential. The later factor is particularly important in operation of cytochrome bc1 as, unlike other mitochondrial complexes, during the catalytic Q
cycle half of the electrons that are transferred from UQH2
to cytochrome c must be “pushed” against the existing
transmembrane electric field (this phenomenon is a result of
bifurcation of electron transfer in the Qo site upon oxidation of UQH2 and spatial arrangement of the heme cofactors in relation to the vector of membrane potential as described in sect. VI).
In mitochondria, under most physiologic conditions, the
energy available to cytochrome bc1 released upon transfer
of electrons from UQH2 to cytochrome c is used to do work
A
ADP
H+
H+
on proton transport across the membrane (this will be referred in the remaining text as the “forward mode” of operation of cytochrome bc1). This occurs when oxygen and
reducing equivalents from the tricarboxylic acid cycle are
continuously supplied while ATP is continuously used up
by the cellular processes (FIGURE 4A). However, as the
membrane potential increases, the energy may become insufficient and the electron flow from the Q pool to the C
pool slows down. At conditions of high proton electrochemical gradient and high degree of reduction of the C
pool, the electron flow through cytochrome bc1 can be reversed (“reverse mode” of operation) (FIGURE 4B). This was
shown by classic experiments in which artificially added
ATP stimulated reverse electron flow in mitochondria (46).
This phenomenon was also observed in experiments with
cytochrome bc1 reconstituted into lipid bilayer (174). In
addition, the reverse electron transfer within cytochrome
bc1 induced by electric field gradient generated by photosynthetic reaction center was observed in membranes of
purple bacteria (233). In fact, the growth of some acidophilic chemolitotrophic bacteria at low pH on ferrous iron
ATP
H+
H+
–
UQH2
UQ
Q pool
UQH2
Δψ
UQ
UQH2
UQH2
UQH2
+
Qi Reduction
Qo Oxidation
cyt. c Reduction
H+
H+
H+
H+
H+
C pool
B
ADP
ATP
H+
UQ
UQ
H+
UQ
Q pool
UQ
Qi Oxidation
Qo Reduction
cyt. c Oxidation
Δψ
UQH2
UQ
–
FIGURE 4. Two possible net directions of
electron and proton transport catalyzed by
cytochrome bc1. A: forward mode: in the
presence of low or moderate membrane
potential and low ATP/ADP ratio, cytochrome bc1 transports protons from matrix to intermembrane space while electrons are transferred from the Q pool to the
C pool. The flow of protons back to matrix
through ATPase results in ATP synthesis.
B: reverse mode: under high membrane
potential and high ATP/ADP ratio, the enzymes work in the opposite direction. Cytochrome bc1 transfers electrons from the C
pool to the Q pool, while ATPase hydrolyzes
ATP to ADP.
UQ
H+
H+
H+
C pool
+
H+
H+
H+
H+
H+
H+
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MARCIN SAREWICZ AND ARTUR OSYCZKA
as a source of electrons depends on cytochrome bc1 that
catalyzes the oxidation of cytochrome c from the C pool
and reduction of UQ from the Q pool (38, 88). The UQH2
is then used to reduce NAD, which is required for CO2
fixation. This peculiar reverse electron transfer from cytochrome c is a consequence of mediocre reducing power of
Fe3⫹/Fe2⫹ couple and the fact that Em of UQ/UQH2 at low
pH at the periplasmic site is much more positive than at the
cytoplasmic site where pH is close to neutral.
The electron recirculation part works on the basis of
action of the two quinone binding sites that together
form a redox loop (161, 178) that plays a role of a “machine” translocating protons across the membrane. The
UQ reduction site (Qi or Qn) is located at the matrix side
of the inner mitochondrial membrane, while the UQH2
oxidation site (Qo or Qp) is located towards the intermembrane space (in this case the terms reduction and
oxidation in the names of the sites refer to the forward
mode of operation of cytochrome bc1). The Qo and Qi
sites are connected electronically by the b-type hemes
located in between. The Qo site is also connected electronically with the C pool by two cofactors: Rieske type
iron-sulfur [2Fe-2S] cluster (FeS) and c-type heme (heme
c1) (FIGURE 5B). This connection constitutes the second
functional part of the enzyme.
VI. COFACTOR CHAINS OF CYTOCHROME
bc1 AS A FRAME FOR THE Q CYCLE
Understanding of the catalytic Q cycle of cytochrome bc1
may appear difficult to a nonspecialist at first contact. In
our opinion, it is a consequence of the fact that a complete
enzymatic cycle of cytochrome bc1 involves two cycles in
which two UQH2 are oxidized and two cytochrome c molecules are reduced while one UQ is reduced (64). This gives
net oxidation of one UQH2 per reduction of two cytochrome c molecules. Such reaction must involve a recirculation of half of the electrons that derive from the Q pool
back to the Q pool (36). The catalytic cycle can thus be
understood as a product of a cooperation of two functional
parts. The first part recirculates electrons from and to the Q
pool, while the second part steers the electrons to the C pool
powering the electron recirculation (FIGURE 5A).
A
The cofactors that build the first and the second part are
commonly referred as a low-potential chain (or b-chain)
and a high-potential chain (or c-chain), respectively. These
terms reflect the difference in Em of the cofactors in the chains:
Em of hemes b span from ⫺160 to ⫹100 mV, while Em of FeS
and heme c1 in high-potential chain is in the range between
⫹200 to ⫹400 mV (69). The redox properties of cofactors
appear to fit properties of the substrate. This can be inferred
from the observations that some bacteria operating on lowpotential menaquinones or living in alkaline environment
B
Qi site
7Å
12.2Å
Q pool
14Å
Heme bL
e–
b chain
Heme bH
Qo site
C pool
23Å
b-state
c-state
9.5Å
>23Å
Heme c1
C
D
Supernumerary
subunits
H+
UQH2
UQ
Q pool
e–
UQH2
UQ
c chain
Rieske
cluster
e–
FIGURE 5. Major functional principles
and structural elements of cytochrome
bc1. A: when the enzyme steers electrons
from the Q pool to the C pool, half of the
electrons are recycled back to the Q pool.
B: spatial arrangement and distances between cofactors and catalytic sites in the
dimer. C: subunit composition of mitochondrial cytochrome bc1 with colormarked three catalytic subunits of one
monomer. D: scheme of electron and proton transfers within the monomer in the
forward mode. Dotted lines in B and D
denote large-scale movement of the FeS
head domain harboring Rieske cluster between b- and c-states.
e–
2H+
Iron-sulfur
protein
226
Cytochrome b
Cytochrome c1
C pool
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REGULATION AND FUNCTION OF CYTOCHROME bc1 IN MITOCHONDRIA
have downshifted redox potential of FeS below ⫹200 mV
(162, 227).
through the c-chain (FeS and heme c1 to reach heme c of
diffusible cytochrome c), while the other passes through
the b-chain (heme bL and heme bH across the membrane
to reach the occupant of the Qi site) (FIGURE 5D). This
reaction is often referred to as a bifurcation of electron
paths and is a central feature of the catalytic cycle critical
for the energetic efficiency of the enzyme. The enzyme
must somehow ensure that the two electrons deriving
from UQH2 do not end up traveling in the same chain nor
the energetically favorable electron transfer from the
low-potential b-chain components to the high-potential
c-chain components takes place (37, 189, 199, 200).
Those types of reactions dissipate energy and are often
referred as short-circuits (FIGURE 6).
All redox cofactors and the catalytic sites are embedded
within the three subunits that are part of the catalytic core
of cytochrome bc1: transmembrane cytochrome b harbors
two hemes b (bL and bH) and provides environment for
quinone binding Qo and Qi sites, membrane-anchored cytochrome c1 harbors heme c1, the membrane-anchored FeS
subunit contains FeS (FIGURE 5C) (128, 132, 269, 277).
Some bacterial cytochromes bc1 (for example in Rhodobacter capsulatus and Paracoccus denitrificans) can just be
composed of these three subunits (21, 142, 216, 272).
Other cytochromes bc1 may have accessory subunits, which
number varies from one in bacterial cytochrome bc1 (for
example, in Rhodobacter sphaeroides) (6, 273) to eight in
mitochondrial cytochrome bc1 (19, 268). These subunits do
not contain redox cofactors and are not involved in energy
conversion. It is suggested that some accessory subunits
may serve as processing peptidase for subunits of complex
III, at least for the FeS subunit. They may also play a role in
positioning the FeS subunit within the complex (19).
The Qo-site-mediated bifurcation delivers one electron to
the Qi site at the time. As full reduction of UQ requires two
electrons, the compound that is created in the Qi site after
delivery of one electron from the Qo site depends on
whether UQ or USQ⫺ occupies the Qi site. If UQ occupies
the Qi site, it is half-reduced to USQ⫺, but if USQ⫺ is
present, it is reduced to UQH2. This way two cycles of the
Qo site are needed to fully reduce UQ to UQH2 at the Qi
site, explaining the overall stoichiometry of one UQH2 molecule per two UQ molecules formed (64).
The arrangement of cofactors in the c- and b-chains creates
a unique set of conditions for redox reactions at the Qo site:
upon two-electron UQH2 oxidation, one electron passes
A
forward
heme bH
C
D
heme bL
Rieske
cluster
UQH2
1
1
2
2
UQH2
1
3
3'
O2-
O2
B
E
reverse
UQH2
2
2'
O2
O2-
F
2
UQ
2
UQ
1
2
2'
O2
1
1
UQ
3
O2-
3'
O2
O2-
FIGURE 6. Energy-conservation, short-circuiting, and ROS generation in the Qo site. A: forward mode:
complete oxidation of UQH2. B: reverse mode: complete reduction of UQ. Blocking reaction 2 in A or B leads
to semiforward or semireverse reaction, respectively, resulting in the formation of USQo. C–F: possible
engagement of short-circuit reactions or leaks of electrons on oxygen when USQo is formed in semiforward or
semireverse reaction (CD or EF, respectively). Numbers in A–F denote the order of electron transfer. Reactions 2= and 3= are alternatives to 2 and 3, respectively, denoting possible competition between superoxide
generation and short-circuits.
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MARCIN SAREWICZ AND ARTUR OSYCZKA
X-ray crystal structures of known cytochrome bc1 complexes showed close positioning of heme bL and bH in the
b-chain that promote fast electron transfer (21, 93, 128,
132, 142, 268, 277). At the same time, the crystals revealed
that the position of the head domain of the FeS subunit
varied. This cofactor was found very close to the Qo site (in
“b-state,” where it can exchange electrons with quinone in
the Qo site), close to heme c1 (in “c-state,” where it can
exchange electrons with heme c1) (FIGURE 5B) or at various
positions between these two states (19, 60, 277). This implicated that the change in the position of FeS head domain
between these two states was needed to transfer electrons
from the Qo site to cytochrome c1. Such a large-scale movement of the FeS head domain is now supported by many
kinetic and biochemical studies (40, 70 –73, 222, 250, 251).
These studies allowed the description of several conformational states where the movement is affected by mutations
and, unlike in the native system, becomes rate-limiting for
electron transfer. There is an ongoing debate (22) as to
whether the movement of the FeS head domain is controlled
by specific conformational changes in the region of substrate binding (94, 268) or by allostery (50, 52, 55, 187) or
whether the changes between the b- and c-state are rather
stochastic (49, 60, 199).
Crystal structures also revealed that the FeS center, despite
the motion, never approaches closer than 23 Å from heme
bL (FIGURE 5B). This provides a simple way of separating
the high- and low-potential chains by slowing direct electron transfer from heme bL to the FeS center beyond the
catalytic timescale. The exact position of quinone within
this 23 Å gap is unknown because quinone has never been
resolved in the Qo site. Crystal structures including the Qo
site inhibitors show two overlapping loci of binding at
which quinone redox catalysis may occur: one inhibitor,
stigmatellin, approaches within ⬃7 Å of FeS (22, 128) (FIGURE 7), while the other, myxothiazol, approaches within
⬃6 Å of heme bL (95). Regardless of the exact position of
quinone in the Qo site, its presence as a redox-active cofactor bridges the 23 Å gap between heme bL and FeS with two
connections, FeS-quinone and quinone-heme bL, each of
less than 14 Å, suitable for fast electron transfer (FIGURE 7).
This creates a potential way for short-circuiting the b-chain
with the c-chain (FIGURE 6). While it is clear that the shortcircuits must be avoided, the molecular mechanism of how
this is accomplished remains unclear (43, 63, 199, 200).
The crystal structures revealed that cytochrome bc1, unlike
other complexes of mitochondrial respiratory chain, is a
homodimer. The two monomeric units assemble so that the
FeS head domain interacting with cytochrome b and cytochrome c1 of one monomer has its membranous anchor
associated with the other monomer. Dimeric nature of cytochrome bc1 was further confirmed in biochemical studies
(270). Interestingly, two hemes bL in the dimer are in a close
distance (14 Å), implicating that electronic connection between monomers via heme bL-bL electron transfer is possible (FIGURE 5B). All other distances between cofactors of
the two monomers are large enough to prevent catalytically
relevant electron transfer between them.
VII. INTERACTIONS OF QUINONE AND
CYTOCHROME c WITH CYTOCHROME
bc1
A. Quinone Binding Qo Site
Despite the fact that the general idea of electronic bifurcation in the Qo site is widely accepted and supported by
numerous biochemical studies, the consequences of this
phenomenon make the understanding of the principles of
the Qo site engineering difficult. Furthermore, some of the
key structural and kinetic elements of catalysis remain obscure. First of all, the lack of crystal structures containing
quinone bound, at any redox state, in the Qo site leaves a
FIGURE 7. Structural elements of the Qo site with bound
inhibitor, stigmatellin. Three major regions in the pocket
probed by mutagenic studies are shown in blue, green, and
magenta. Green rings denote histidines coordinating Rieske
cluster. Residues E272 (on cytochrome b subunit shown in
gray) and H161 (on FeS subunit shown in amber) form hydrogen bonds with the inhibitor. Dashed lines show distances
between chroman ring and cofactors: heme bL and Rieske
cluster.
228
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REGULATION AND FUNCTION OF CYTOCHROME bc1 IN MITOCHONDRIA
high degree of freedom in constructing models of the binding, electron transfers, and proton release into the intermembrane space. The second important aspect is associated
with the experimental difficulty in isolating intermediate
states of the catalysis, which also leaves a high degree of
freedom for mechanistic considerations. The first report of
detection of semiquinone intermediate associated with the
Qo site (75a) was questioned (136), and until recently (44,
223, 261, 276), no other intermediates have been reported.
This general difficulty in detecting semiquinone in the Qo
site (USQo) has been considered as confirmatory to Mitchell’s original idea that this species must be highly unstable
(177). This became the basis for constructing thermodynamic properties of USQo (43, 79, 275).
Comparative analysis of various crystal structures containing different inhibitors of the Qo site identified the
region of quinone binding consistent in amino acid composition with earlier mapping by site-directed mutagenesis (39, 59). In addition, it showed displacements of
polypeptide backbone in the region expected for the quinone binding (94, 268).
The most recognized representatives of the inhibitors of the
Qo site (262) are stigmatellin and myxothiazol. The former
inhibitor was found to form a hydrogen bond between hydroxyl group of the chroman ring and one of the histidines
coordinating Rieske cluster (128, 164). In this way, stigmatellin creates conditions in which the FeS head domain is in
the closest position to the Qo site. On the basis of this
observation, it is assumed that quinone binds to the Qo site
in virtually the same conformation as stigmatellin (62, 164,
165). On the other hand, myxothiazol binds closer to the
heme bL not in direct contact with the FeS head domain.
The two different niches for inhibitor binding suggest that
there is enough degree of freedom for quinone to move
within the site (62), or for two quinones to bind simultaneously but with different affinity as proposed in the double
occupancy model (79, 80). In fact, the idea of dynamics of
quinone within the site was exploited by some models of
UQH2 oxidation and also can be considered as one of the
explanations for lack of the recognized electron density of
quinone in the Qo site in crystal structures. In addition,
several biochemical studies reported that stoichiometry of
the binding can be larger than 1 quinone per 1 Qo site,
which would seem consistent with relatively spacious binding pocket (16, 36, 79, 80, 232). On the other hand, recent
molecular dynamic simulations indicated that there is not
enough room in the cavity to accommodate more than one
molecule of substrate (207).
Extensive site-directed mutagenesis systematically explored
structural and functional elements of the Qo site. The mutations examined so far appear to come mainly from three
regions of cytochrome b subunit (FIGURE 7): helix C, helix
cd, and proximity to the conserved PEWY motif on the ef
loop (23, 39, 111). These mutations can have various effects
on phenotype, assembly of the whole complex, measured
rates of electron transfer, enzymatic activity, quinone binding, and occupancy of the Qo site (11, 78, 79, 175, 225,
226). The mutations that affect assembly of the complex
almost inevitably lead to the loss of function (226). On the
other hand, within the group of the existing Qo site mutations that allow a proper assembly of the complex, only a
few mutations fully abolish the functionality of cytochrome
bc1 (23, 39, 111). A number of positions can be mutated
with little or no effects, while the energetic efficiency of
cytochrome bc1 is retained at levels that support growth of
the cells under conditions that require the presence of functional cytochrome bc1. Although in some cases sensitivity to
the type of the introduced by mutation amino acid side
chain can be observed, many positions appear to tolerate
many types of side chain change. In general, this indicates
that the Qo site pocket may have a rather significant structural flexibility and a lot of engineering tolerances for a
mutational change. Interestingly, this flexibility is in part
explained by recent molecular simulations indicating that
benzoquinone moiety of quinone does not directly interact
with the side chains of amino acids of cytochrome b that
create the binding pocket. Instead, it may interact with atoms of the polypeptide main chain through the molecules of
water (207). This would explain why defining specific quinone binding motif is difficult (100).
Because quinone at the Qo site is flanked by two redox
centers, each coming from a different chain of cofactors,
there are two possible general reaction schemes of twoelectron UQH2 oxidation or UQ reduction that can be considered. The “sequential” scheme considers two separate
one-electron reactions occurring sequentially one after the
other (37, 62, 165, 189, 199). If UQH2 is to be oxidized
(forward mode), the first reaction is a reduction of FeS by
UQH2 leading to generation of USQo intermediate while
the second reaction is a reduction of heme bL by USQo
leading to formation of UQ (completing the oxidation of
UQH2) (FIGURE 6A). If UQ is to be reduced (reverse mode),
the first reaction is reduction of UQ to USQo by heme bL
followed by the reduction of USQo to UQH2 by FeS (FIGURE
6B). The “concerted” scheme assumes that the two oneelectron reactions involving FeS and heme bL required for
oxidation of UQH2 or reduction of UQ occur simultaneously with no USQo intermediate formed during this reaction (129, 199, 236, 253, 280). The concerted scheme became a subject of considerations for two main reasons. It
helped explain difficulty in detecting USQo intermediate. It
also provided a built-in mechanism for prevention of unwanted short-circuit reactions (199, 200). This is because
the concerted scheme assumes that one-electron reactions
of the sequential scheme are not allowed, eliminating the
short-circuits that are the result of these one-electron reactions occurring in “inappropriate” order (FIGURE 6, C–F).
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MARCIN SAREWICZ AND ARTUR OSYCZKA
There is still no consensus which scheme best describes
reaction mechanism at the Qo site. Recent reports of detection of USQo seem to point towards the sequential scheme.
In two cases, small amounts of USQo were identified by
electron paramagnetic resonance (EPR) spectroscopy as
typical radical signal of semiquinones (44, 276). The low
levels of detected USQo were consistent with the concept of
high instability of this intermediate. In another study, however, two populations of USQo were identified (223). The
first population was represented by a radical signal similar
to the other semiquinones but, unlike in previous reports
(44, 75a, 276), was strongly affected by interaction with
metal cofactors of the Qo site (reminiscent of its presence in
the site). Intriguingly, the second population was recognized as a semiquinone spin-coupled to reduced Rieske cluster generating a new spectroscopic identity. The relatively
large amount of the new signal suggested that USQo is not
as highly unstable as commonly assumed, at least when
USQo interacts with Rieske cluster. It was proposed that
USQo coupled to Rieske cluster might represent an initial
state of oxidation of UQH2 when oxidized FeS is withdrawing electron from UQH2 (223).
Regardless of properties of all so far detected USQo, its
presence in the site according to the sequential scheme
requires additional level of control to prevent short-circuits. Mechanism of such control remains unknown and
is subject of various proposals (43, 63, 186, 200, 215)
that all introduce additional complexity to the system. In
this context, the built-in mechanism for short-circuit prevention in a concerted scheme still remains an attractive
alternative.
When discussing sequential versus concerted scheme, one
should bear in mind that a clear distinction between these
two mechanisms is not as obvious as intuition might tell.
The problem concerns relative timescales of processes. One
could ask what is the minimum time separation between the
first and the second electron transfer to consider the reaction as “virtually concerted.” Kinetically, the concerted
term means that time separation between the two electron
transfer steps is shorter than time required for significant
atomic rearrangement (femtoseconds) (199).
An important aspect of operation of the Qo site is rapid
reversibility of electron transfers between the substrate and
FeS and heme bL. At the same time, biochemical studies
indicate that quinone bound at this site freely equilibrates
with the Q pool (79). This, in the context of reversibility,
may have important consequences on the mechanism of the
Qo site catalysis. The molecule of quinone present in the site
makes connection between the b- and c-chains allowing
rapid exchange of electrons in both directions (199). This
means that the net electron flow (forward or reverse) is a
consequence of an evolution of the site into the new state
that does not allow the reaction to be reversed, for example,
230
by replacing the substrate at the site (49). It follows that the
redox state of the Q pool may actively influence the direction of electron flow through the site.
Proton transfers at the Qo site remain largely unknown.
Using the stigmatellin binding mode as a reflection of quinone binding in the catalytically active state, most of the
proposed scenarios assume that upon UQH2 oxidation the
first proton coming from UQH2 is used to protonate His161 (one of the ligand of the Rieske cluster), while the
second proton is used to protonate Glu-272 (Glu-295 in
bacteria) in PEWY motif (62, 129, 186, 204). Those two
residues seem best suited as initial proton acceptors in crystal structures (FIGURE 7) given that they form hydrogen
bonds with stigmatellin bound at the Qo site (130). However, until now, apart from the observation that histidine is
the redox-linked protonation site of Rieske cluster (131),
there is no direct experimental evidence for any of the proton reactions at the Qo site. Mutating His-161 (His-156 in
bacteria) is not informative in this case as it disrupts assembly of the whole FeS subunit (74). On the other hand, mutating Glu-272 into nonprotonable amino acids does not
abolish enzymatic activity of cytochrome bc1, indicating
that if this residue is involved in proton transfers, it is not
the only possible proton acceptor in the site (157, 201, 230,
264). As with other quinone oxidation/reduction sites, the
pathways for proton appear relatively insensitive to perturbations (202). Another alternative, indicated by recent MD
simulations, is that water molecules can act as direct acceptors of both protons from UQH2 (207).
B. Quinone Binding Qi Site
The Qi site is a terminal part of the low-potential chain that
catalyzes two-electron reduction of UQ to UQH2 when cytochrome bc1 works in a forward mode. This reaction results from two sequential steps in which electrons are delivered from the same cofactor chain. Such reaction requires
stabilization of USQ intermediate formed in the site after
every second UQH2 oxidation cycle of the Qo site. Indeed,
the stable semiquinone originating from cytochrome bc1,
and as we now know being the semiquinone bound in the Qi
site (USQi), was detected even before the formulation of the
Q cycle (265). The possibility to detect USQi intermediate
greatly facilitated structural and functional studies on the
Qi site. Additionally, the crystal structures of cytochrome
bc1 containing UQ molecule in this site provided direct and
detailed information on the quinone binding (126, 128).
The fact that UQ can be crystallized in the Qi site, but not in
the Qo site, indicates that the binding mode at these two
sites is different and the binding affinity for the Qi site is
larger. Equilibrium redox titrations suggested that UQH2
binding to the Qi site is slightly stronger than UQ from the
Q pool, while the binding constant for USQi must be several
orders of magnitude larger than that for UQ (217).
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REGULATION AND FUNCTION OF CYTOCHROME bc1 IN MITOCHONDRIA
Mutagenic studies and EPR spectroscopy identified conservative residues responsible for binding and stabilization of
quinone and USQi (76, 112, 120, 143). One of them is
His-201 (His-217 in bacteria) which appears to stabilize
USQi through the H-bonding of ⬎C⫽O group to imidazole
ring of histidine. Other important residues include Asp-228
(Asp-252 in bacteria) and Ser-205 (Asn-221). Quinone
molecule binds very closely to heme bH with the edge-toedge distance between heme moiety and benzoquinone ring
of ⬃6 Å. Such close distance allows for rapid electron transfer from reduced heme bH to UQ (or USQi). Intriguingly, in
a closely related cytochrome b6f, the position of quinone is
occupied by an additional heme ci (or cx) not present in
cytochrome bc1, while quinone is shifted further away from
heme bH (149, 242).
As the complete reduction of UQ requires an uptake of two
protons from matrix, the reaction scheme can be divided
into following steps (143): UQ ⫹ bH2⫹ ↔ USQi⫺ ⫹ bH3⫹
(1) and USQi⫺ ⫹ bH2⫹ ⫹ 2H⫹ ↔ UQH2 ⫹ bH3⫹ (2).
Despite the lack of experimental data on the electron-associated proton transfer, it can be envisaged that two protons
are attached to the benzoquinone ring in response to the
appearance of the second electron. This scheme is supported by the observation that USQi is in anionic form
(USQi⫺) (76, 143). Crystal structures revealed two regions
rich in protonable groups of amino acid side chains and
water molecule paths for consideration as proton paths
linking the catalytic site with the membrane surface (129,
153). Interestingly, the proton transfer is suggested to engage polar groups of cardiolipin as this lipid has been
shown to bind in proximity to one of the putative proton
channels (9, 129, 153, 208).
Considering the reversibility of reactions, USQi⫺ can originate either from the reaction (1) or reversing the reaction
(2). Nevertheless, USQi⫺ detected in experiments appear to
come from reversing reaction (2), as it is present along with
reduced heme bH. It is important to bear in mind that the
reverse reaction (2) fills b-chain with electrons, which in
consequence may slow down the rate of oxidation of UQH2
at the Qo site. This effect can be compared with the effect of
the weak competitive inhibitor of the Qi site. As a result, at
high range of UQH2 concentrations, the activity of cytochrome bc1 may decrease (29, 35). This “downregulation”
may play an important role in controlling the electron flux
through the respiratory chain.
Although the crystal structures do not show any significant structural changes caused by binding of antimycin
(126), a potent inhibitor of the Qi site, biochemical and
spectroscopic studies have implicated that it might influence the average position of FeS head domain in the
complex (51, 222, 260). While this long-range structural
effect has been exploited by various models that propose
allostery within the dimer of cytochrome bc1 (50, 52–
54), the origin and the time domain of this inhibitorinduced effect are unknown.
C. Cytochrome c Binding Site
Cytochrome bc1 interacts with soluble cytochrome c at the
solvent-exposed surface of cytochrome c1 subunit. The interaction regions of both cytochrome c and c1 surround the
area where hemes are exposed. Consequently, the association of the two proteins allows two hemes to attain distances in the range of 10 Å (152), which is short enough that
fast electron exchange between them becomes possible.
It is generally accepted that interactions between proteins
involve long-range electrostatic interactions that facilitate
formation of an encounter complex, while short-range interactions foster the stabilization of a tight complex in a
proper spatial configuration (197, 228, 231, 257). Electrostatic forces are considered to be a dominant factor that
contributes to binding of cytochrome c to cytochrome bc1,
which is inferred from a significant salt dependence of electron transfer between these proteins (90, 114, 121, 135,
249). An importance of short-range hydrophobic interactions between surfaces of cytochrome c1 subunit and cytochrome c was proposed on the basis of X-ray structures of
cytochrome bc1 co-crystallized with its redox partner (152,
238).
Extensive chemical modifications and mutagenic studies
identified crucial amino acid residues that stabilize the complex of the proteins through interactions of negatively and
positively charged residues on the surfaces of cytochrome c1
and c, respectively (30, 163, 241, 249). The transient binding of cytochrome c to cytochrome bc1 in solution was
inferred from the analysis of electron transfer between these
proteins (175) and from the measurements based on the
techniques that were independent of electron transfer, such
as plasmon wave-guide resonance spectroscopy (75) or EPR
(206, 221, 224).
The complexes are relatively long-lived at low ionic
strength, but their lifetime decreases rapidly with an increase of salt concentration. At ionic strength typical of
physiological conditions, the complexes are very short-lived
and the stationary concentration of bound cytochrome c is
low. This implicates a mechanism in which under physiological conditions cytochrome c does not form stable, longlived complexes but rather constantly collides with the surface of cytochrome bc1 and electron transfer takes place
coincidentally with one of such collision (117, 206, 221).
VIII. GENERATION OF REACTIVE OXYGEN
SPECIES
Discussion on generation of ROS by cytochrome bc1 started
in the mid 1970s when submitochondrial particles inhibited
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MARCIN SAREWICZ AND ARTUR OSYCZKA
with antimycin and fueled with succinate were able to produce a significant amount of superoxide anion (31, 32,
195). The superoxide production was subsequently linked
to redox reactions of the Qo site (42, 123, 147, 190, 255).
Consistent with that notion, current, widely shared view
considers highly unstable USQo as a direct one-electron
donor for oxygen (43). This is envisaged on the basis of very
low potential of USQo/UQ couple (less than ⫺400 mV; Ref.
200) and relatively high redox potential of O2⫺/O2 couple
(approximately ⫺160 mV in aqueous solution at pH 7; Ref.
266). While there are some suggestions that USQi may also
contribute to ROS production (210), this much more stable
semiquinone is not generally considered as a source of electrons that can leak on oxygen. Exclusion of the Qi site from
ROS generation activity was supported by the fact that ROS
from cytochrome bc1 are observed when inhibitor antimycin is bound to the Qi site. Furthermore, a complete inhibition of the Qo site by stigmatellin, which still allows formation of USQi (through reverse reaction), leads to a complete
loss of superoxide generation activity (189). It is likely that
stabilization of USQi through the H-bonding of ⬎C⫽O
group to imidazole ring of histidine can protect USQi
against the reaction with oxygen (259).
In reversibly operating Qo site, USQo can be formed in two
ways (29, 82, 145, 189, 209, 220): as a part of forward
reaction, when FeS withdraws electron from UQH2 and the
second electron transfer from USQo to heme bL does not
take place, or as a part of reverse reaction when heme bL
donates an electron to UQ and the second electron transfer
from FeS to USQo does not take place. These two reactions
can be referred to as “semiforward” and “semireverse,”
respectively (FIGURE 6).
Currently both semiforward and semireverse schemes are
considered as possible reactions leading to USQo that can
interact with oxygen. Both reaction schemes require as an
initiation a reduced state of heme bL, which is consistent
with the observation that ROS are observed when the electron transfer between heme bL and heme bH is impeded by
blocking the Qi site with antimycin or by increase in membrane potential (144, 218). In the semiforward scheme, reduced heme bL is unable to take electron from USQo and
thus cannot convert it to UQ (103, 145, 189, 209). In the
semireverse scheme, the reduced heme bL initiates a formation of USQo (FIGURE 6) (29, 82, 220). As the semireverse
reaction, unlike the semiforward reaction, uses UQ as a
substrate, the recognition that the semireverse reaction may
lead to ROS production came from analysis of a dependence of the rate of ROS production in submitochondrial
particles and in isolated complexes on the redox state of the
Q pool. The maximum rate of superoxide generation was
detected when the Q pool was partly oxidized, which is
consistent with the semireverse rather than semiforward
mechanism (82), for which the rate of superoxide generation should increase monotonically with reduction level of
232
the Q pool. An impact of the Q pool redox state is also
inferred from the stimulating effect of complex II inhibitors
on ROS generation by cytochrome bc1 (82, 84, 168, 240).
Further support for the semireverse scheme came from the
analysis of various cofactor knockout mutants of cytochrome bc1 (29). With these studies the model was further
developed to describe an important constraint associated
with the kinetic effects of the motion of the FeS head domain. The model proposes that probability of ROS generation increases as the distance, separating the FeS head domain from the Qo site at the exact time of USQo formation,
increases (29, 220). This is because the positions of FeS
remote from the Qo site diminish a chance that FeS engages
in electron transfer to interact with USQo (either by reducing USQo to UQH2 which would complete the reverse reaction, or oxidizing USQo to UQ which would complete the
short-circuit reaction) before USQo reacts with oxygen.
From the kinetic point of view, this can be ascribed as a
competition taking place on timescale of seconds (i.e., timescale beyond the catalytic timescale) between the leaks of
electrons on oxygen and the short-circuit reactions (FIGURE
6). When the short-circuits become effective (FeS is in or
close to the Qo site), the leaks are diminished, and vice
versa. This way, the short-circuits emerge as not only unwanted reactions that should be avoided at all costs as they
dissipate energy, but in some cases they may be beneficial in
helping to diminish or modulate the leaks of single electrons
and the associated ROS levels. Interestingly, the notion
about a “beneficial” role of short-circuits develops also for
other bioenergetics enzymes (219). In closely related cytochrome b6f (this enzyme is part of photosynthetic chains in
plants and catalyzes electron transfer from plastoquinol to
plastocyanin) (57), the short-circuits were proposed to be
important as an “emergency exit” pathway to bypass the
Q-cycle in the enzyme with disabled Qi site to sustain the
function of the entire photosynthetic chain (169).
It should be noted that although the reaction schemes based
on unstable USQo assume its high reactivity with oxygen,
this reaction has never been directly confirmed experimentally. In this context, the possibility that other cofactors,
such as low-potential heme bL, directly reduce oxygen cannot be ruled out. Interestingly, recent studies indicate that
there might be states of USQo that are not highly reactive
with oxygen (223). These include a state in which USQo is
spin-coupled to reduced Rieske cluster. This state is observed in the presence of oxygen in particular in the FeS
motion mutants that eliminate superoxide activity of cytochrome bc1 (29, 220).
In general, the level of ROS production by mitochondria appears to strongly depend on respiration state (2, 192). It is
rather a common misconception that with faster respiration
rate, the conversion of oxygen to superoxide should increase.
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REGULATION AND FUNCTION OF CYTOCHROME bc1 IN MITOCHONDRIA
However, it was observed that factors limiting the rate of
oxygen consumption (respiration rate) favor superoxide production. Typically, this can occur under high membrane potential, nearly inactive ATPase, high NADH:NAD⫹ ratio, or
hypoxia (2, 118, 144, 192, 237). Under those conditions, the
reduction levels of individual cofactors in respiratory complexes increase. This, in view of the kinetic constraints inherent to the proposed mechanisms of superoxide generation by
the Qo site, explains why complex III can be one of the contributors to ROS generation under those types of conditions.
return back to respiratory chain via reaction of superoxide
anion with oxidized cytochrome c (205, 279). Because of
this reaction, cytochrome c is considered an important element of antioxidative system of mitochondria.
IX. H-SHAPED ELECTRON TRANSFER
SYSTEM IN DIMER AND ITS
PHYSIOLOGICAL IMPLICATIONS
In most textbooks, the principles of Q cycle are described
considering cytochrome bc1 monomer, which has its justification in the fact that each monomer is equipped with all
structural elements necessary to complete the catalytic cycle. All of these were described above: quinone binding Qo,
and Qi sites and cytochrome c binding site linked together
by two cofactor chains. However, the dimeric structure of
this enzyme invoked several intriguing mechanistic issues
that became a subject of intense debate.
From all components of mitochondrial electron transport
chain that are considered as possible contributors to mitochondrial ROS (complex I, cytochrome bc1, and recently
also complex II), only cytochrome bc1 is an enzyme that
appears to release most of superoxide to the intermembrane
space (34, 123, 239), as expected considering the localization of the Qo site (FIGURE 8). This topology of ROS release
from the Qo site is proposed to have implications for redox
signaling (26, 84). Generated superoxide after rapid dismutation to hydrogen peroxide may target components of the
antioxidative system both in the intermembrane space and
in cytosol (98). In this reaction scheme, electrons irreversibly leak from the respiratory chain due to dismutation of
superoxide. In an alternative pathway, electrons on superoxide molecules present in the intermembrane space may
Matrix
The most fundamental question concerns the electronic connection between the monomers, which given the distances between the cofactors in dimeric structure, is possible at the level
of hemes bL. While the existence of this connection has been
considered since the first crystal structures of cytochrome bc1
were solved (50, 55, 61, 110, 199, 234, 269), the experimental
Cytochrome bc1
–
TQ
Δψ
3
O2
e–
4
1
UQ
TOC
+
Activation
Regulation
Signaling
UQH2
UQH2
O2–
H2O2
Reduced
e–
Cyt. c
selfexchange
superoxide
dismutase
2
Oxidized
O2
Intermembrane space
H2O
Complex IV
FIGURE 8. Regulation and ROS-generating activity of cytochrome bc1. Factors influencing the electron
transfer between the Q and C pools catalyzed by cytochrome bc1 include the following: 1 and 2, redox state of
the Q and C pools, respectively; 3, magnitude of the membrane potential; 4, constituents of the membrane
that can interact with quinone binding sites: tocopherol (TOC) and tocoquinone (TQ). Under some conditions,
Qo site may become source of superoxide which is released to intermembrane space. Lines denote the
following: dotted black, electron transfer paths; solid black, diffusion of O2 and H2O; solid red, diffusion of
⫺
and H2O2); dashed green, participation of ROS in activation,
cytochrome c; solid green, diffusion of ROS (O2
regulation, and/or signaling; solid yellow, diffusion of TOC and TQ.
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233
MARCIN SAREWICZ AND ARTUR OSYCZKA
evidence for it was not available until recently. It came from
mutagenic studies demonstrating that the derivatives of the
enzyme with inactivated Qo site in one monomer and Qi site in
the other (cross-inactivated forms) are able to support enzymatic turnover using effectively the heme bL-bL electron transfer as the only available connection linking the functional Qo
and Qi sites (67, 68, 150, 244). The electron transfer through
this two-heme bridge occurs in a millisecond timescale, which
falls within the timescale of the catalytic turnover of native
enzyme. It was also observed that these cross-inactivated forms
of cytochrome bc1 are able to sustain growth of the bacterial cells
under conditions that obligatorily require presence of functional
cytochrome bc1 (87a, 150, 151). This indicates that enzyme relying just on the paths involving heme bL-bL electron transfer performs the bioenergetic function in vivo.
The at least millisecond timescale of electron transfer between two hemes bL converts the cofactor chains in the
dimer into an H-shaped electron transfer system connecting
all four quinone catalytic sites (FIGURES 5B AND 9). A basic
operational principle of this system is that it enables any
connection between catalytic sites of the opposite sides of
the membrane to be enzymatically competent. The physiological meaning of this design remains an open issue.
With our current knowledge on the thermodynamics of the
reactions in cytochrome bc1 taken into account, it can be
anticipated that electron transfer between hemes bL and bH
of each monomer is favored over heme bL-bL electron transfer as long as there are no factors that influence the electron
transfer route (234). The factor inherently present and dynamically changing in respiring mitochondria is membrane
potential, which when increases, impedes the electron
transfer from heme bL to heme bH, but at the same time it
may increase the fraction of electrons that travel through
the hemes bL-bL bridge (234). Thus the membrane potential
comes as one of the prominent factors that can modulate
the inter- versus intramonomer electron transfer.
The presence of the intermonomer connection creates a possibility of dismutation of two semiquinones that may occupy
quinone binding sites. This way it might be possible to fully
reduce one UQ molecule in one of the Qi sites using two electrons, each delivered from a different Qo site. This could provide
physiological advantage for the cells as it may enhance efficiency
of the Q cycle under shortage of UQH2 in the Q pool.
Another possible physiological advantage of the H-shaped
electron transfer system is related to the effects of accumulation of mitochondrial damage. This process is related to a
relatively high frequency of mutations within mitochondrial DNA (mtDNA) and a possibility that the mutations
occurring in genes coding for subunits of respiratory enzymes affect operation of the enzymes expressed from these
genes (13, 17, 77, 99, 173, 254). It is generally believed that
in the process of mitochondrial damage, mutations in
234
mtDNA progressively accumulate in time, which may be
inherent to aging and/or progress of development of several
mitochondria-related diseases (148). For cytochrome bc1,
this concerns cytochrome b subunit which is coded by a
gene located in mtDNA. Given that this gene does not differentiate between monomers and that mtDNA is present in
several copies in mitochondria, the presence of mutations
in just a part of the copies of the gene is likely to result in
expression of both symmetrically and asymmetrically mutated dimers of cytochrome bc1 (FIGURE 9) as observed in
bacterial two-plasmid model systems (45, 141, 151). Thus
it can be anticipated that the amount of the asymmetrically
damaged complexes increases with age. In this context, the
H-shaped electron transfer provides a clear advantage as it
builds in redundancy to allow physiological function of the
enzyme even after operational damage of one part.
Yet another possible physiological advantage of the Hshaped electron transfer system is related to the ROS generation by cytochrome bc1. All current models of ROS generation consider the prolonged state of reduced heme bL as
condition increasing probability of superoxide generation
by the Qo site (29, 43, 82, 83, 145, 189, 209, 220). Such a
state becomes more frequent when the electron outflow
from the Qi site is blocked or suppressed by inhibition or
mutation. If such damage or inhibition occurs asymmetrically in a dimer (is present only in one monomer), the connection between monomers helps remove electrons from
hemes in the b-chain, decreasing the chance of superoxide
formation by the enzyme. Considering that cytochrome bc1
evolved in an anoxygenic environment, it is not clear whether
possible protective role of geometric proximity of two hemes
bL against ROS was a coincidental effect or an evolutionarilydriven design. Nevertheless, one cannot exclude that some
tuning of energy levels might have occurred during evolution
(219), for example, to minimize the probability of direct reaction of reduced heme bL with oxygen.
X. COMPOUNDS THAT INFLUENCE THE
ACTIVITY OF CYTOCHROME BC1
So far, many chemical compounds have been recognized as
efficient modulators of cytochrome bc1 activity. These are
not only naturally occurring inhibitors and synthetic drugs
or pesticides but also some substances that are an inherent
part of the cellular environment like vitamin E and its metabolites or certain lipids. Typically, these compounds target the catalytic Qo or Qi sites by competing for binding
with natural substrates.
The very strong inhibitors that tightly bind to the catalytic
sites are considered as toxic for cells as they significantly
restrict or even prevent the electron transfer through complex III leading to disruption of mitochondrial respiration.
Some of these compounds (such as myxothiazol, stigmatellin, or antimycin) (262, 247, 248) are naturally produced
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REGULATION AND FUNCTION OF CYTOCHROME bc1 IN MITOCHONDRIA
Mutated
mtDNA
Crossinactivated
Mutated
mtDNA
Asymmetricallymutated
functional
Unmutated
mtDNA
ROS
Qi
FIGURE 9. H-shaped electron transfer
system of cytochrome bc1 and consequences of symmetric and asymmetric mutation patterns in the context of mitochondrial damage. The system connects electronically all four catalytic sites of the dimer,
and only symmetrically inactivated enzymes
with interrupted connection of both Qo sites
with both Qi sites are nonfunctional.
Qi
ROS
Qo
Qo
Symmetricallymutated nonfunctional
Unmutated
cytochrome bc1
by certain bacteria. Many Qo site inhibitors (such as strobilurin derivatives or famoxadone) are widely used in agriculture as pesticides because they are efficient fungicides but
are, at the same time, relatively less toxic for mammals.
Other applications include medicine. For example, the synthetic inhibitor of the Qo site atovaquone is used in combination with other drugs to treat parasite diseases, in particular malaria, babesia, and toxoplasmosis (12, 140, 193).
The potential use of cytochrome bc1 inhibitors as drugs
stimulates research to seek new compounds that would effectively target cells of protozoa parasites (15, 127, 278).
The weak inhibitors that reversibly bind to the catalytic
sites are potential regulators of cytochrome bc1 activity.
One such compound is vitamin E (tocopherol) and the
products of its degradation (tocoquinones). It appears that
vitamin E has not only antioxidant properties but can also
modify the activity of cytochrome bc1 (108, 109, 113, 188,
274). There are also observations that vitamin E can influence ROS production by mitochondria, and it is proposed
that this effect results from the interference of vitamin E
with electron transfer at the level of the Qo site (66, 188)
(FIGURE 8). The concept of binding of vitamin E to the Qo
site is supported by the fact that this compound shares the
same chroman ring as stigmatellin.
Interestingly, ␣-tocoquinone appears to act as an alternative substrate for the Qi site where it can be reduced to
␣-tocopheryl hydroquinone (109) (FIGURE 8). Because ␣-tocopheryl hydroquinone cannot be oxidized at the Qo site,
this reaction partially uncouples the Q cycle. On the other
hand, it is proposed that this reaction may be beneficial,
especially under oxidative stress, as ␣-tocopheryl hydroquinone is a compound of antioxidative properties.
It remains to be seen how the weakly bound external compounds can modulate the activity of cytochrome bc1 in the
context of its dimeric function and its possible role in regulation of respiration on the molecular level. It also remains to be
seen how and why they modulate ROS production. The regulatory function of these compounds may be of great importance for tissues under oxidative stress and aging.
XI. CONCLUSIONS
Cytochrome bc1 is one of the key enzymes of mitochondrial
respiratory chain, where it plays a major role as proton
translocating machinery utilizing the energy stored in the
difference in redox potentials between the membrane pool
of quinone (Q pool) and water-soluble pool of cytochrome
c (C pool). Several features of this homodimeric enzyme
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MARCIN SAREWICZ AND ARTUR OSYCZKA
make it a potential point of regulation that dynamically
modulates the electron flux through the mitochondrial respiratory chain in response to the actual physiological conditions. Our goal was to point out those features and discuss them in the context of the molecular mechanism of
cytochrome bc1 operation and physiological functioning of
mitochondria (FIGURE 8).
First, each monomer builds in two quinone binding sites
where two opposite redox reactions take place. This means
that the substrate for one site is the product of the second
site, and vice versa. These sites are located on the opposite
sides of the membrane and are electronically connected
through the chain of cofactors providing a path for reversible exchange of electrons between the sites. In this system
the direction of electron flow depends on rates of all partial
reversible reactions within the cofactor chains, including
the exchange of UQ/UQH2 within the catalytic sites. Overall, this makes the activity of the whole complex sensitive to
redox state of the Q pool. In addition, as the enzyme connects the Q pool with C pool, it may switch between the
forward and the reverse mode of operation in response to
the changes in bioenergetic state of the respiratory chain
(FIGURE 4).
Second, the electronic connection between monomers of the
dimer links functionally all four quinone catalytic sites,
forming the H-shaped electron transfer system which increases the number of available connections between the
sites. In this system, any connection between the two sites
on the opposite sides of the membrane is enzymatically
competent (FIGURE 9). The H-shaped electron transfer system can provide physiological advantage for the cells as it
may enhance the efficiency of the enzymatic cycle under
shortage of UQH2 in the pool. It also builds in redundancy
to allow physiological function of the enzyme even after
operational damage of one part. This may be advantageous
in the context of mitochondrial mutations that accumulate
upon aging or under oxidative stress.
Third, in addition to the energy-conserving reactions, cytochrome bc1 under certain conditions generates superoxide
which, unlike in the case of other respiratory complexes, is
released to the intermembrane space of mitochondria. This
reaction results in electron leakage diminishing proton
pumping efficiency of the enzyme. However, because of its
location, superoxide generated by cytochrome bc1 can be
considered as a potential factor engaged in the redox signaling (FIGURE 8). The role of this compound in sensing the
redox state of mitochondria is conceivable given that the
level of superoxide generation strongly depends on the reduction levels of cofactors in cytochrome bc1 which, in
turn, change in response to various factors including the
redox state of the Q pool and the C pool and the magnitude
of membrane potential. In fact, the exact amounts of superoxide released by cytochrome bc1 may be under redox and
236
kinetic control given that this reaction appears to compete
with other electron transfer reactions that take place within
the enzyme. One type of those reactions includes the shortcircuits that, at cost of dissipating energy, retain single electrons within the enzyme preventing their leakage (FIGURE
6). The other type of reactions is inherent to a uniting action
of the H-shaped electron transfer system which can collect
and neutralize single electrons generated by cytochrome bc1
to diminish ROS.
Fourth, some of the constituents naturally present in mitochondrial membrane may modulate the activity of cytochrome bc1 or ROS production by interfering with quinones in binding to the catalytic sites (FIGURE 8). Also,
some metabolites of vitamin E can serve as an alternative
substrate which undergoes reduction at quinone-reducing site of cytochrome bc1. On one hand, these types of
reactions interfere with the catalytic cycle; on the other
hand, they become a support for antioxidative system in
membranes.
At present, considering all the structural and functional
details of the quinone binding catalytic sites and the cofactor chains of cytochrome bc1, understanding of the operation of cytochrome bc1 is at the advanced stage of molecular
characterization. Nevertheless, several crucial aspects of
operation of this enzyme still remain elusive. For example,
at the molecular level, further progress in description of
intermediates at the quinone oxidation site and reactions
leading to ROS generation is needed. At the cellular level,
the potential role of ROS generated by cytochrome bc1 in
signaling requires further studies. Undoubtedly, as our
thinking and methodology advance to better link physics/
chemistry with physiology, these masterpieces of bioenergetic machinery will excite us with new discoveries at all
levels.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: A.
Osyczka, Dept. of Molecular Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian
University, ul. Gronostajowa 7, 30-387 Kraków, Poland
(e-mail: artur.osyczka@uj.edu.pl).
GRANTS
This work was supported by The Wellcome Trust International Senior Research Fellowship (to A. Osyczka).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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