ELECTRON TRANSPORT CHAIN AND OXIDATIVE PHOSPHORYLATION SEMESTER IV, PAPER CC8

ELECTRON TRANSPORT CHAIN & OXIDATIVE PHOSPHORYLATION

The NADH and FADH2 formed in glycolysis, fatty acid oxidation, and the citric acid cycle are
energy-rich molecules because each contains a pair of electrons having a high transfer potential.
When these electrons are used to reduce molecular oxygen to water, a large amount of free
energy is liberated, which can be used to generate ATP. Oxidative phosphorylation is the process
in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O2 by a
series of electron carriers. This process, which takes place in mitochondria, is the major source of
ATP in aerobic organisms. The oxidation of fuels and the phosphorylation of ADP are coupled
by a proton gradient across the inner mitochondrial membrane.

The Electron Transport Chain:

Electrons are transferred from NADH to O 2 through a chain of three large protein complexes
called NADH-Q oxidoreductase, Q-cytochrome c oxido-reductase, and cytochrome c oxidase.
Electron flow within these transmembrane complexes leads to the transport of protons across
the inner mitochondrial membrane.

Figure: Summary of the flow of electrons and protons through the four complexes of the respiratory chain.

Complex I:
Complex I, also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase, is a large
enzyme composed of 42 different polypeptide chains, including an FMN-containing flavoprotein
and at least six iron sulfur centers. High-resolution electron microscopy shows Complex I to be
L-shaped, with one arm of the L in the membrane and the other extending into the matrix. The
reaction catalyzed by this enzyme appears to be

NADH + H+ + Q → NAD+ + QH2

The initial step is the binding of NADH and the transfer of its two high-potential electrons to the
flavin mononucleotide (FMN) prosthetic group of this complex to give the reduced form,
FMNH2. Electrons are then transferred from FMNH 2 to a series of iron-sulfur clusters, the
second type of prosthetic group in NADH-Q oxidoreductase. Fe-S clusters in iron-sulfur proteins
(also called non-heme iron proteins) play a critical role in a wide range of reduction reactions in
biological systems. NADH-Q oxidoreductase contains both 2Fe-2S and 4Fe-4S clusters. Iron
ions in these Fe-S complexes cycle between Fe 2+ (reduced) or Fe 3+(oxidized) states. Electrons in

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the iron-sulfur clusters of NADH-Q oxido-reductase are shuttled to coenzyme Q. The flow of
two electrons from NADH to coenzyme Q through NADH-Q oxido-reductase leads to the
pumping of four hydrogen ions out of the matrix of the mitochondrion. NADH binds to a site on
the vertical arm and transfers its electrons to FMN. These electrons flow within the vertical unit
to three 4Fe-4S centers and then to a bound Q. The reduction of Q toQH 2 results in the uptake of
two protons from the matrix. The pair of electrons on bound QH 2 is transferred to a 4Fe-4S
center and the protons are released on the cytosolic side. Finally, these elections are transferred
to a mobile Q in the hydrophobic core of the membrane, resulting in the uptake of two additional
protons from the matrix.

Figure: NADH:ubiquinone oxidoreductase

(Complex I).

Complex II:
Although smaller and simpler than Complex I, it contains five prosthetic groups of two types
and four different protein subunits. Subunits C and D are integral membrane proteins, each
with three transmembrane helices. They contain a heme group, heme b, and a binding site for
ubiquinone, the final electron acceptor in the reaction catalyzed by Complex II. Subunits A and
B extend into the matrix, they contain three 2Fe-2S centers, bound FAD, and a binding site for
the substrate, succinate. The path of electron transfer from the succinate-binding site to FAD,
then through the Fe-S centers to the Q-binding site, is more than 40 Å long, but none of the
individual electron-transfer distances exceeds about 11 Å—a reasonable distance for rapid
electron transfer.

The citric acid cycle enzyme succinate dehydrogenase, which generates FADH2 with the
oxidation of succinate to fumarate, is part of the succinate-Q reductase complex (Complex II), an
integral membrane protein of the inner mitochondrial membrane. Two other enzymes, glycerol
phosphate dehydrogenase and fatty acyl CoA dehydrogenase, likewise transfer their high
potential electrons from FADH 2 to Q to form ubiquinol (QH 2), the reduced state of ubiquinone.
The succinate- Q reductase complex and other enzymes that transfer electrons from FADH 2 to Q,
in contrast with NADH-Q oxido-reductase, do not transport protons. Consequently, less ATP is
formed from the oxidation of FADH 2 than from NADH.

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Figure: Succinate-Q reductase (Complex II).

Complex III & the Q-cycle:

The second of the three proton pumps in the respiratory chain is Q-cytochrome c oxido-reductase
(also known as Complex III and cytochrome reductase). A cytochrome is an electron-transferring
protein that contains a heme prosthetic group. The iron ion of a cytochrome alternates between a
reduced ferrous (+2) state and an oxidized ferric ( +3) state during electron transport. The function
of Q-cytochrome c oxido-reductase is to catalyze the transfer of electrons from QH 2 to oxidized
cytochrome c (cyt c), a water-soluble protein, and concomitantly pump protons out of the
mitochondrial matrix.

Q-cytochrome c oxidoreductase is a dimer with each monomer containing 11 subunits. Q

cytochrome c oxidoreductase itself contains a total of three hemes, contained within two
cytochrome subunits: two b-type hemes, termed heme b L (L for low affinity) and heme b H (H
for high affinity), within cytochrome b, and one c-type heme within cytochrome c 1. In addition
to the hemes, the enzyme also contains an iron-sulfur protein with an 2Fe-2S center. This center
is termed the Rieske center. Finally, Q-cytochrome c oxidoreductase contains two distinct
binding sites for ubiquinone termed Qo and Qi, with the Qi site lying closer to the inside of the
matrix.

The Q- cycle:
The mechanism for the coupling of electron transfer from Q to cytochrome c to
transmembrane proton transport is known as the Q cycle. The cycle begins as ubiquinol (QH 2)
binds in the Qo site. Ubiquinol transfers its electrons, one at a time. One electron flows first to
the Rieske 2Fe-2S cluster, then to cytochrome c1, and finally to a molecule of oxidized
cytochrome c, converting it into its reduced form. The reduced cytochrome c molecule is free to
diffuse away from the enzyme. The second electron is transferred first to cytochrome b L, then to
cytochrome b H , and finally to an oxidized uniquinone bound in the Qi site. This quinone (Q)

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molecule is reduced to a semiquinone anion (Q · -). Importantly, as the QH2 in the Qo site is
oxidized to Q, its protons are released to the cytosolic side of the membrane. This Q molecule in
the Qo site is free to diffuse out into the ubiquinone pool.

At this point, Q · - resides in the Qi site. A second molecule of QH 2 binds to the Qo site and
reacts in the same way as the first. One of its electrons is transferred through the Rieske center
and cytochrome c 1 to reduce a second molecule of cytochrome c. The other electron goes
through cytochromes b L and b H to Q · - bound in the Qi site. On the addition of the second
electron, this quinone radical anion takes up two protons from the matrix side to form QH 2. The
removal of these two protons from the matrix contributes to the formation of the proton gradient.
At the end of the Q cycle, two molecules of QH2 are oxidized to form two molecules of Q, and
one molecule of Q is reduced to QH 2, two molecules of cytochrome c are reduced, four protons
are released on the cytoplasmic side, and two protons are removed from the mitochondrial
matrix.

Figure: Q cycle (Complex III).

Complex IV: The final stage of the electron-transport chain is the oxidation of the reduced
cytochrome c generated by Complex III, which is coupled to the reduction of O 2 to two
molecules of H2O. This reaction is catalyzed by cytochrome c oxidase (Complex IV). It consists
of 13 subunits, of which 3 (called subunits I, II, and III) are encoded by the mitochondrial
genome. Cytochrome c oxidase contains two heme A groups and three copper ions, arranged as
two copper centers, designated A and B. One center, CuA /CuA , contains two copper ions linked
by two bridging cysteine residues. This center initially accepts electrons from reduced
cytochrome c. The remaining copper ion, CuB, is coordinated by three histidine residues. The
two heme A molecules, termed heme a and heme a3 , have distinct properties because they are
located in different environments within cytochrome c oxidase. Heme a functions to carry
electrons from CuA/CuA , whereas heme a3 passes electrons to CuB, to which it is directly
adjacent. Together, heme a3 and CuB form the active center at which O 2 is reduced to H2O.

The catalytic cycle begins with the enzyme in its fully oxidized form. One molecule of reduced
cytochrome c transfers an electron, initially to CuA/CuA. From there, the electron moves to heme
a, then to heme a3, and finally to CuB , which is reduced from the Cu2+ (cupric) form to the Cu+
(cuprous) form. A second molecule of cytochrome c introduces a second electron that flows

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down the same path, stopping at heme a3, which is reduced to the Fe 2+ form. Recall that the iron
in hemoglobin is in the Fe 2+ form when it binds oxygen. Thus, at this stage, cytochrome c
oxidase is poised to bind oxygen and does so. The proximity of CuB in its reduced form to the
heme a3-oxygen complex allows the oxygen to be reduced to peroxide (O 22-), which forms a
bridge between the Fe 3+ in heme a3 and CuB2+. The addition of a third electron from cytochrome
c as well as a proton results in the cleavage of the O-O bond, yielding a ferryl group, Fe 4+ = O,
at heme a3 and CuB2+-OH. The addition of the final electron from cytochrome c and a second
proton reduces the ferryl group to Fe 3+-OH. Reaction with two additional protons allows the
release of two molecules of water and resets the enzyme to its initial, fully oxidized form.

This reaction can be summarized as:

Cytochrome c oxidase evolved to pump four additional protons from the matrix to the
cytoplasmic side of the membrane in the course of each reaction cycle for a total of eight protons
removed from the matrix.

Figure: Cytochrome Oxidase Mechanism (Complex IV).

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OXIDATIVE PHOSPHORYLATION:

The electrochemical energy inherent in the difference in proton concentration and separation of
charge represents a temporary conservation of much of the energy of electron transfer. The
energy stored in such a gradient is termed the proton-motive force. According to the model, the
proton-motive force drives the synthesis of ATP as protons flow passively back into the matrix
through a proton pore associated with ATP synthase. Later it was revealed that enzyme-bound
ATP forms readily in the absence of a proton-motive force; the role of the proton gradient is not
to form ATP but to release it from the synthase.

Structure of ATP synthase:

It is a large, complex membrane-embedded enzyme that looks like a ball on a stick. The 85-Å
diameter ball, called the F1 subunit, protrudes into the mitochondrial matrix and contains the
catalytic activity of the synthase. In fact, isolated F1 subunits display ATPase activity. The F1
subunit consists of five types of polypeptide chains -  3, 3 , ,  , and The and subunits,
which make up the bulk of the F1, are arranged alternately in a hexameric ring; they are
homologous to one another and are members of the P-loop NTPase family. Both bind
nucleotides but only the subunits participate directly in catalysis. The central stalk consists of
two proteins: and . The subunit includes a long -helical coiled coil that extends into the
center of the  3 3 hexamer. The subunit breaks the symmetry of the  33 hexamer: each of the
subunits is distinct by virtue of its interaction with a different face of . The F0 subunit is a
hydrophobic segment that spans the inner mitochondrial membrane. F0 contains the proton
channel of the complex. This channel consists of a ring comprising from 10 to 14 c subunits that
are embedded in the membrane. A single a subunit binds to the outside of this ring. The proton
channel depends on both the a subunit and the c ring. The F0 and F1 subunits are connected in
two ways, by the central stalk and by an exterior column. The exterior column consists of one
a subunit, two b subunits, and the subunit. Hence, the structure can be considered as: (1) a
moving unit, or rotor, consisting of the c ring and the stalk, and (2) a stationary unit, or stator,
composed of the remainder of the molecule.

Figure: ATP Synthase (Complex V).

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Paul Boyer proposed a binding-change mechanism for proton-driven ATP synthesis. This
proposal states that changes in the properties of the three subunits allow sequential ADP and Pi
binding, ATP synthesis, and ATP release. Interactions with the subunit make the three 
subunits in equivalent. One subunit can be in the T, or tight, conformation. This conformation
binds ATP with great avidity, i.e., it has a high affinity for converting ADP and Pi to ATP and
does not easily release ATP. A second subunit will then be in the L, or loose, conformation. This
conformation binds ADP and P i. It too, is sufficiently constrained that it cannot release bound
nucleotides. The final subunit will be in the O, or open, form. This form can exist with a bound
nucleotide in a structure that is similar to those of the T and L forms, but it can also convert to
form a more open conformation and release a bound nucleotide.

The interconversion of these three forms can be driven by rotation of the subunit. Suppose the
subunit is rotated 120 degrees in a counterclockwise direction (as viewed from the top). This
rotation will change the subunit in the T conformation into the O conformation, allowing the
subunit to release the ATP that has been formed within it. The subunit in the L conformation will
be converted into the T conformation, allowing the transition of bound ADP + Pi into ATP.
Finally, the subunit in the O conformation will be converted into the L conformation, trapping
the bound ADP and Pi so that they cannot escape. The binding of ADP and Pi to the subunit now
in the O conformation completes the cycle. This mechanism suggests that ATP can be
synthesized by driving the rotation of the subunit in the appropriate direction.

The direct observation of rotary motion of the subunit is strong evidence for the rotational
mechanism for ATP synthesis. The mechanism depends on the structures of the a and c subunits
of F0.

Figure: Binding Change Model of ATP Synthase

ATP-ADP Shuttle:

The major function of oxidative phosphorylation is to generate ATP from ADP. However, ATP
and ADP do not diffuse freely across the inner mitochondrial membrane.A specific transport
protein, ATP-ADP translocase (also called adenine nucleotide translocase or ANT), enables
these molecules to traverse this permeability barrier. Most important, the flows of ATP and ADP
are coupled. ADP enters the mitochondrial matrix only if ATP exits, and vice versa. The reaction
catalyzed by the translocase, which acts as an antiporter, is

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ATP-ADP translocase is highly abundant, constituting about 14% of the protein in the inner
mitochondrial membrane. The translocase, a dimer of identical 30-kd subunits, contains a single
nucleotide-binding site that alternately faces the matrix and cytosolic sides of the membrane.
ATP and ADP (both devoid of Mg2+) are bound with nearly the same affinity. In the presence of
a positive membrane potential (as would be the case for an actively respiring mitochondrion), the
rate of binding site eversion from the matrix to the cytosolic side is more rapid for ATP than for
ADP because ATP has one more negative charge. Hence, ATP is transported out of the matrix
about 30 times as rapidly as is ADP, which leads to a higher phosphoryl transfer potential on the
cytosolic side than on the matrix side. The translocase does not evert at an appreciable rate
unless a molecule of ADP is bound at the open, cytos olic site, which then everts to the
mitochondrial matrix side. This feature ensures that the entry of ADP into the matrix is precisely
coupled to the exit of ATP. The other side of the coin is that the membrane potential and hence
the proton-motive force are decreased by the exchange of ATP for ADP, which results in a net
transfer of one negative charge out of the matrix. ATP-ADP exchange is energetically expensive;
about a quarter of the energy yield from electron transfer by the respiratory chain is consumed to
regenerate the membrane potential that is tapped by this exchange process. The inhibition of this
process leads to the subsequent inhibition of cellular respiration as well.

Fig: ATP-ADP Shuttle

Mitochondrial transporters:

ATP-ADP translocase is but one of many mitochondrial transporters for ions and charged
metabolites. The phosphate carrier, which works in concert with ATP-ADP translocase, mediates
the electroneutral exchange of H 2PO4- for OH- (or, indistinguishably, the electroneutral symport
of H2PO4- and H+). The combined action of these two transporters leads to the exchange of
cytosolic ADP and P i for matrix ATP at the cost of an influx of one H+. The dicarboxylate
carrier enables malate, succinate, and fumarate to be exported from mitochondria in exchange for
Pi. The tricarboxylate carrier transports citrate and H + in exchange for malate. Pyruvate in the
cytosol enters the mitochondrial matrix in exchange for OH- (or together with H+) by means of

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the pyruvate carrier. These mitochondrial transporters and more than five others have a common
structural motif. They are constructed from three tandem repeats of a 100-residue module, each
containing two putative transmembrane segments.

Fig: Mitochondrial Transporters

BLOCKERS OF ELECTRON TRANSPORT CHAIN

PROTON MOTIVE FORCE AND ATP SYNTHESIS

Much of the energy is used to pump protons out of the matrix. For each pair of electrons
transferred to O2,

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four protons are pumped out by Complex I, four by Complex III, and two by Complex IV. The
electrochemical energy inherent in this difference in proton concentration and separation of
charge represents
a temporary conservation of much of the energy of electron transfer. The energy stored in such a
gradient,
termed the proton-motive force, has two components: (1) the chemical potential energy due to
the
difference in concentration of a chemical species (H 2) in the two regions separated by the
membrane, and (2)
the electrical potential energy that results from the separation of charge when a proton moves
across the
membrane without a counterion. When protons flow spontaneously down their electrochemical
gradient, energy is made available to dowork. In mitochondria, chloroplasts, and aerobic
bacteria, the electrochemical energy in the proton gradient drives the synthesis of ATP from
ADP and Pi. The chemiosmotic model, proposed by Peter Mitchell, is the paradigm for this
mechanism. According to the model the electrochemical energy inherent in the difference in
proton concentration and separation of charge across the inner mitochondrial membrane—the
proton-motive force—drives the synthesis of ATP as protons flow passively back into the matrix
through a proton pore associated with ATP synthase.

Mitchell used “chemiosmotic” to describe enzymatic reactions that involve, simultaneously, a

chemical reaction
and a transport process. Because the energy of substrate oxidation drives ATP synthesis in
mitochondria, we would expect inhibitors of the passage of electrons to O2 (such as cyanide,
carbon monoxide, and antimycin A) to block
ATP synthesis. More surprising is the finding that the converse is also true: inhibition of ATP
synthesis
blocks electron transfer in intact mitochondria. This obligatory coupling can be demonstrated in
isolated
mitochondria by providing O2 and oxidizable substrates, but not ADP. Under these conditions,
no ATP synthesis can occur and electron transfer to O 2 does not proceed. Coupling of oxidation
and phosphorylation can also be demonstrated using oligomycin or venturicidin, toxic antibiotics
that bind to the ATP synthase in mitochondria. These compounds are potent inhibitors of both
ATP synthesis and the transfer of electrons through the chain of carriers to O2. Because
oligomycin is known to interact not directly with the electron carriers but with ATP synthase, it
follows that electron transfer and ATP synthesis are obligately coupled; neither reaction occurs
without the other. Chemiosmotic theory readily explains the dependence of electron transfer on
ATP synthesis in mitochondria.

Uncoupled Mitochondria in Brown Fat Produce Heat

The uncoupling of oxidative phosphorylation is a means of generating heat to maintain body

temperature in hibernating animals, in some newborn animals (including human beings), and in
mammals adapted to cold. Brown adipose tissue, which is very rich in mitochondria (often
referred to as brown fat mitochondria), is specialized for this process of nonshivering
thermogenesis. The inner mitochondrial membrane of these mitochondria contains a large

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amount of uncoupling protein (UCP), here UCP-1, or thermogenin, a dimer of 33-kd subunits
that resembles ATP-ADP translocase. UCP-1 forms a pathway for the flow of protons from the
cytosol to the matrix. In essence, UCP-1 generates heat by short-circuiting the mitochondrial
proton battery.

Most newborn mammals, including humans, have a type of adipose tissue called brown fat in
which fuel oxidation serves not to produce ATP but to generate heat to keep the newborn warm.
This specialized adipose tissue is brown because of the presence of large numbers of
mitochondria and thus large amounts of As a result of this short-circuiting of protons, the energy
of oxidation is not conserved by ATP formation but is dissipated as heat, which contributes to
maintaining the body temperature of the newborn.

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