Inhibitors of the Mitochondrial

Electron Transport Chain

Karim Tabbaa

GMS 6201 Basic Medical

Biochemistry

10/12/2010
 Introduction

Inhibition of the mitochondrial electron transport chain by certain inhibitory drugs

can have serious consequences for the normal energy production of a cell. The inhibition
of mitochondrial electron transport leads to a breakdown in the proton gradient across the
inner mitochondrial membrane, thereby hindering ATP production by the ATP synthase
enzyme. Compounds that inhibit the mitochondrial electron transport chain pose a serious
challenge to normal cellular activity and can induce cell death by autophagy or by
apoptosis, depending on the inhibitor. Some positive medical applications of
mitochondrial electron transport chain inhibitors include inducing cell death in harmful
mutated or cancer cells. This paper seeks to detail inhibitors of electron transport and
discuss the medical applications and implications of those inhibitors. This paper also
outlines the biochemical processes involved in the normal activity of the mitochondrial
electron transport chain (Hwan Han et al).

Adenosine triphosphate (ATP) serves as the main source of energy for cell
survival. ATP can be derived by the cell from biological sources of energy such as
carbohydrates, fats and proteins using cellular metabolism mechanisms. In eukaryotic
cells, the bulk of oxidative cellular metabolism forming ATP occurs in the mitochondria.
Most ATP synthesis occurs during oxidative phosphorylation in the mitochondria, when a
proton gradient formed from the electron transport chain drives the formation of ATP from
ADP via the ATP synthase enzyme. The reactions of the electron transport chain occur in
an ordered series in the inner membrane of the mitochondria and involve energy carriers
NADH and FADH2 produced from glycolysis and the Krebs cycle. The series of reactions
in the inner membrane of the mitochondria occur at various protein complexes and are
coupled with H+ ejections into the inner membrane space of the mitochondria. The
ejection of H+ to the intermembrane space forms a proton gradient used to facilitate
production of ATP by oxidative phosphorylation using ATP synthase enzyme (King)
(Brookes).

Background on Mitochondrial Electron Transport

The mitochondrial electron transport assembly consists of a chain of protein

complexes located in the inner mitochondrial membrane that catalyzes oxidation
reduction reactions in a series. Coenzyme energy carriers NADH and FADH2, products
from the Citric Acid Cycle, are pumped from the mitochondrial matrix to the inner
mitochondrial membrane, where they are involved in the electron transport chain
oxidation reduction reactions. Electrons enter the transport chain from cytosolic NADH to
mitochondrial NADH but can also be supplied by the glycerol phosphate shuttle. In the
course of the reactions, NADH is oxidized by a series of catalytic redox carriers that are
integral proteins of the inner mitochondrial membrane. The reactions are very exergonic
and are coupled to a transport process whereby the protons from NADH are translocated
to a space between the inner and outer mitochondrial membrane known as the
intermembrane space. The transport of protons into the intermembrane space leads to
the formation of a proton gradient across the inner membrane of the mitochondria (King)
(Brookes).

The mitochondrial electron transport chain consists of four different complexes

clustered in a series, known as Complex I, II, III, and IV. Complex I, known as NADH-
CoQ oxidoreductase, comprises NADH dehydrogenase, flavin mononucleotide (FMN)
prosthetic group as cofactor, and non-heme iron proteins containing one sulfur center. In
Complex I, NADH transfers electrons to ubiquinone (CoQ), whereby NADH binds to
Complex I and transfers two electrons to the FMN prosthetic group and then the electrons
are transferred to iron-sulfur proteins (FeS) in Complex I (See Figure I). The two electrons
from the reduced FeS proteins are then transferred to ubiquinone (CoQ) along with two
protons. Ubiquinone thus becomes reduced to the ubiquinol (CoQH2) while the FeS
proteins are oxidized back to Fe 3+. Ubiquinol is mobile, small and lipid soluble, allowing it
to diffuse easily in the mitochondrial membrane and shuttle electrons to Complex III. See
Figure 1. (Brookes) (King).

Figure I

Complex II contains the enzyme succinate dehydrogenase catalyzing the oxidation

of succinate to fumarate coupled with the reduction of ubiquinone to ubiquinol (CoQ to
CoQH2). CoQH2 donates its electrons to Complex III. Complex III consists of heme
proteins known as cytochromes b and c1 and non-heme iron sulfur (FeS) proteins. The
cytochromes contain heme iron (Fe), which becomes reduced in the course of the
reaction. CoQH2 passes two electrons to cytochrome b, causing Fe3+ to be reduced to
Fe2+. The electrons are passed to the FeS protein and then to cytochrome c 1. From
cytochrome c 1, the electrons are transferred to cytochrome c. Cytochrome c is a small
and mobile protein that accepts electrons from Complex III and shuttles them to Complex
IV, which serves as the last electron transport chain protein (Brookes) (King).

See Figure II-IV for illustrations:

Figure II

Figure III

Figure IV
 Complex IV, known as cytochrome c oxidase, comprises heme proteins
cytochrome a and cytochrome a3, which both use iron (Fe) and cooper (Cu) to handle to
electrons transferred to them from cytochrome c. Four cytochrome c molecules transfer
four electrons to Complex IV, with the copper molecules becoming oxidized. The
electrons are then transferred to 4 H+ molecules and an O2 molecule to form two (H2O)
water molecules. Oxygen acts as the final electron acceptor in the electron transport
chain, being reduced and forming water molecules. Electron transfer of the electron
transport chain in the mitochondrial matrix is coupled with H+ ejection from the matrix into
the intermembrane space; creating a proton gradient that powers ATP synthase. ATP is
generated from the proton gradient that forms in the intermembrane space of the
mitochondria when NADH and FADH2 are oxidized during the transfer of electrons via the
electron transport chain. When NADH becomes oxidized to NAD+, three protons are
pumped into the intermembrane space. When FADH2 becomes oxidized to FAD+ two
protons are pumped, creating an electrochemical gradient with enough potential to
generate three ATP for each NADH oxidized and two ATP for each FADH2 oxidized
(Brookes) (King).

Inhibitors of Mitochondrial Electron Transport

Different inhibitors of the mitochondrial electron transport chain act on different

complexes of the transport chain and trigger effects worth noting. In a study conducted by
Chen et. al, an inhibitor of Complex I known as Rotenone has been determined to induce
autophagy contributing to cell death in cancer cells through mediation of reactive oxygen
species. The same study showed that an inhibitor of Complex II, known as
thenoyltrifluoroacetone (TTFA) has also been determined to induce cell death in cancer
cells through mediation of reactive oxygen species. The study conducted by Chen et. al
showed that in primary mouse astrocytes, Rotenone and TTFA failed to induce
autophagy. The findings suggest that the Complex I and II inhibitors, Rotenone and
TTFA, selectively induce autophagic cell death in cancer cells. These results may have
considerable implications in the medical treatment of cancer patients (Chen, Y. et al).

The biochemical explanation of the Chen et. al study illustrates that Complex I and
II inhibitors, Rotenone and TTFA, induce autophagy and contribute to cell death in the
transformed cell line HEK 293 and in cancer cell lines U87 and HeLa. Cell autophagy
manifests by the formation of autophagosomes and autolysosomes in the cell that
degrade the cell itself. During autophagy, cytoplasmic constituents are combined into
autophagosomes, which fuse with lysosomes to form autolysosomes. Autolysosomes
degrade the cell under such conditions as nutrient starvation. Complex I inhibitor
Rotenone and Complex II inhibitor TTFA significantly increased the formation of
autophagosomes and autolysosomes over a 72 hour period (Chen, Y. et al).
 Within Complex I, the electron transfer from NADH to ubiquinone is coupled to the
transfer of two protons per electron across the inner mitochondrial membrane. Complex I
accounts for a significant portion of the proton trans-locating capacity of the electron
transport chain. Rotenone blocks Complex I near the binding site of ubiquinol, the
electron acceptor of Complex I. The inhibition of Complex I by Rotenone disrupts the
proton gradient needed to generate ATP. Rotenone increases the levels of reactive
oxygen species in mitochondrial particles oxidizing NADH. Rotenone blockage of the
Complex I ubiquinol binding site increases the reduction of the NADH dehydrogenase site
at Complex I and increases electron leak to reactive oxygen species. Elevated
production of reactive oxygen species from Complex I leaks into the mitochondrial matrix
causing mitochondrial damage or leads mitochondrial matrix antioxidants to inactivate the
reactive oxygen species. (Chen, Q. et. al, 2003) (Okun et. al).

It was determined that the cell death in the transformed cell line HEK 293 and in
cancer cell lines U87 and HeLa was mediated by reactive oxygen species production
(Chen et. al). Reactive oxygen species include free oxygen radicals such as hydroxyl
radical (HO), superoxide (O2), and hydrogen peroxide (H2O2), which are generated as by-
products of mitochondrial respiration. Excessive amounts of reactive oxygen species can
be harmful to a cell and can induce cell death. Cells possess antioxidant control systems
which are essential for a cell’s survival. Reactive oxygen species tend to be generated
following the collapse of the mitochondrial electron transport chain. Therefore, when
Complex I or Complex II are inhibited by Rotenone and TTFA respectively, production of
reactive oxygen species is induced in the cell, leading to autophagic cell death. Noting
that cells produce antioxidant enzymes to protect themselves against damage caused by
excessive production of reactive oxygen species, when autophagy proceeds in a cell, it
blocks production of important antioxidants, including catalase, thereby allowing reactive
oxygen species to be unrestricted in the cell and resulting in autophagy mediated by
reactive oxygen species (Hwan Han et. al) (Chen et. al).

The study of the transformed cell line HEK 293 and cancer cell lines U87 and
HeLa indicated that cell death was induced by autophagy and not by apoptosis, when
Rotenone and TTFA inhibitors blocked Complex I and Complex II of the mitochondrial
electron transport chain (Chen et.al). The study further indicated that reactive oxygen
species were responsible for inducing cell death by autophagy because autophagy
blocked the caspase enzyme responsible for apoptosis. With the caspase enzyme
blocked, apoptotic cell death would not be induced in the cell. Blockage of caspase
activation leads to degradation of the important antioxidant, catalase, which leads to
levels of reactive oxygen species rising. The increased levels of reactive oxygen species
in transformed and cancer cell lines undergoing Complex I and Complex II inhibition lead
to autophagic cell death, though the precise mechanism remains unclear beyond that
(Chen, Y. et.al).
Complex III Inhibitor: Antimycin A

Complex III serves as a key site for generation of reactive oxygen species which
can lead to cell death if antioxidant defense mechanisms do not counteract production of
reactive oxygen species. The compound Antimycin A serves as an effective inhibitor of
Complex III, inhibiting the electron flow between cytochrome b and cytochrome c1.
Complex III has two centers where reactive oxygen species can be generated, the Q0
center, oriented in the direction of the intermembrane space, and the Qi center, located in
the inner membrane and oriented in the direction of the mitochondrial matrix (see Figure
A). Inhibition of Complex III by Antimycin occurs at the Qi center and increases
superoxide (O2) production at the Q0 center, directing reactive oxygen species away from
the mitochondrial antioxidant defenses. High levels of reactive oxygen species left
unrestrained can lead to mitochondrial damage and ultimately cell death (Chen, Q. et. al,
2003) (Guidarelli et al).

Figure A

Antimycin A, a Complex III inhibitor and compound derived from bacteria called
Streptomyces kitazawensis has been observed to inhibit succinate and NADH oxidase in
the metabolic cellular respiration process. Results from a study on pulmonary
adenocarcinoma A549 cells suggest that Antimycin A may serve as an effective agent
inducing cell death in pulmonary cancer cells (Hwan Han et. al). In the study of pulmonary
cancer cells, Antimycin A was shown to inhibit the growth of human pulmonary
adenocarcinoma A549 cells by inducing cell cycle arrest as well as triggering apoptosis.
Results from the study suggest that an increase in levels of reactive oxygen species were
correlated with apoptosis in A549 cells, although the exact mechanism was unknown by
researchers (Hwan et. al).

During apoptosis, the collapse of mitochondrial membrane potential occurs. The

collapse of the mitochondrial membrane potential can be caused by an inhibitor of the
mitochondrial electron transport chain. Antimycin A was found to induce the loss of
mitochondrial membrane potential in A549 adenocarcinomic cells, damaging
mitochondrial function and consequently inducing apoptosis. Antimycin A leads to a loss
of mitochondrial membrane potential because it disrupts the oxidation/reduction reactions
in Complex III of the electron transport chain, thereby halting the proton gradient across
the inner mitochondrial membrane. Reactive oxygen species levels, in particular those of
H2O2 and O2, were shown to contribute to Antimycin A induced cell death (Hwan Han et.
al).

Complex IV Inhibitor: Cyanide

Complex IV of the mitochondrial electron transport chain, known as cytochrome c

oxidase, can be inhibited by cyanide. Inhibition of cytochrom c oxidase by cyanide can
result in serious damage to a cell and can lead to cell death. Cyanide is a complex toxic
compound which affects multiple reactions in a cell, the primary being its disruption of
oxidative cellular metabolism by inhibiting cytochrome c oxidase reduction. In a normal
cell with a functioning electron transport chain, cytochrome c oxidase becomes reduced
with the transfer of electrons to the complex from cytochrome c. Cyanide inhibits
cytochrome c from transferring its electrons to cytochrome c oxidase, leading to the
production of elevated levels of reactive oxygen species in the mitochondria, which are
damaging to cells. Cyanide also inhibits important antioxidant defense enzymes in a cell
that serve to protect cells against the harmful effects of reactive oxygen species. Inhibition
of antioxidant mechanisms by cyanide allows reactive oxygen species to affect great
damage to a cell. Acute cyanide poisoning carries serious consequences and its effects
have been linked to delayed neuropathy and likened to Parkinson’s Disease (Leavesley
et. al).

Conclusion

Functioning mitochondrial electron transport chains serve as essential parts of a

complex system that humans have been blessed with, working for the proper functioning
and maintenance of life. Problems in a mitochondrial electron transport chain hold serious
consequences for cells and can lead to cell death. Inhibitors of mitochondrial electron
transport, when used in an effective manner, can be utilized for medical benefit. Current
research seeks to uncover the precise mechanism which Complex I inhibitors, such as
Rotenone, use to target cancer cells. Complex III inhibitors, in particular Antimycin A,
have been used as effective agents in facilitating apoptosis in lung cancer cells. The role
of reactive oxygen species in relation to Complex I and III inhibitors seems to facilitate cell
death in cancer cells, though current research seeks to define a more precise mechanism
for the evident relationship. The role of reactive oxygen species in facilitating cell death
also appears with regards to cyanide poisoning and more details are being discovered
about cyanide poisoning to uncover an antidote in order to reverse its effects.
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