March 2, 2009

Metabolism III –
Oxidative phosphorylation
 Oxidative Phosphorylation

Essence of Oxidative Phosphorylation. Oxidation and ATP synthesis are coupled by

transmembrane proton fluxes.
Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer
of electrons from NADH or FADH2to O2by a series of electron carriers.
 Role of mitochondria

Cytosolic side or P-side

Matrix side or N-side

• Mitochondria contain the respiratory assembly, the enzymes of the citric acid cycle, and
the enzymes of fatty acid oxidation.
• Mitochondria has two membranes.
• Oxidative phosphorylation takes place in the inner mitochondrial membrane as opposed to
most of the reactions of TCA cycle that takes place in the Matrix.
• The outer membrane is quite permeable to most small molecules and ions because it
contains many copies of mitochondrial porin, a 30–35 kd poreforming protein also known as
VDAC, for voltage-dependent anion channel.
• The two faces of this membrane will be referred to as the matrix side and the cytosolic side
(the latter because it is freely accessible to most small molecules in the cytosol). They are also
called the N and P sides, respectively, because the membrane potential is negative on the
matrix side and positive on the cytosolic side.
 The concept of Redox potential
If the reaction proceeds in the direction

the reactions in the half-cells (referred to

as half-reactions or couples) must be

Thus, electrons flow from the sample half-cell to

the standard reference half-cell, and the sample-
cell electrode is taken to be negative with respect
to the standard-cell electrode. The reduction
potential of the X:X-couple is then observed
voltage at the start of the experiment (when X, X-,
and H+ are 1 M). The reduction potential of the
H+:H2couple is defined to be 0 volts.

The meaning of the reduction potential is now evident. A negative reduction potential means
that the reduced form of a substance has lower affinity for electrons than does H 2, as in the
preceding example. A positive reduction potential means that the reduced form of a
substance has higher affinity for electrons than does H 2. These comparisons refer to standard
conditions—namely, 1 M oxidant, 1 M reductant, 1 M H +, and 1 atmosphere H2. Thus, a strong
reducing agent (such as NADH) is poised to donate electrons and has a negative reduction
potential, whereas a strong oxidizing agent (such as O 2) is ready to accept electrons and has
The reduction potentials of many biologically important redox couples are known. It is
like those presented in chemistry texts except that a hydrogen ion concentration of 10-7
M (pH 7) instead of 1 M (pH 0) is the standard state adopted by biochemists. This
difference is denoted by the prime in E´0. Recall that the prime in ΔG°´ denotes a
standard free-energy change at pH 7.
The standard free-energy change ΔG°´ is related to the change in reduction potential
ΔE´0 by

in which n is the number of electrons transferred, F is a proportionality constant called

the faraday [23.06 kcal mol-1 V-1 (96.48 kJ mol-1 V-1)], ΔE´0 is in volts, and ΔG°´ is in
kilocalories or kilojoules per mole.

How can the energy associated with a proton gradient be quantified? The free-energy
change for a species moving from one side of a membrane where it is at concentration c1
to the other side where it is at a concentration c2 is given by in which Z is the electrical
charge of the transported species and ΔV is the potential in volts across the membrane.
Under typical conditions for the inner mitochondrial membrane, the pH outside is 1.4
units lower than inside [corresponding to log10 (c2/c1) of 1.4] and the membrane potential
is 0.14 V, the outside being positive. Because Z = +1 for protons, the free-energy change
is (2.303 × 1.98 × 10-3 kcal mol-1 K-1 × 310 K × 1.4) + ( + 1 × 23.06 kcal mol-1 V-1 × 0.14 V) = 5.2
kcal mol-1 (21.8 kJ mol-1). Thus,
each proton that is transported out
of the matrix to the cytosolic side corresponds to 5.2 kcal
mol-1 of free energy.
Electron transport chain
 Structure of NADH-Q Oxidoreductase

Matrix side

Cytosol side
Structure of NADH-Q Oxidoreductase (Complex I). The structure, determined by
electron microscopy at 22-Å resolution, consists of a membrane-spanning part and a long
arm that extends into the matrix. NADH is oxidized in the arm, and the electrons are
transferred to reduce Q in the membrane.
How is ATP synthesis coupled to Proton gradient?
 Chemiosmotic hypothesis

How is the oxidation of NADH coupled to the phosphorylation of ADP? It was first
suggested that electron transfer leads to the formation of a covalent high-energy
intermediate that serves as a high phosphoryl transfer potential compound or to the
formation of an activated protein conformation, which then drives ATP synthesis. The
search for such intermediates for several decades proved fruitless.

Testing the Chemiosmotic Hypothesis of

Peter Mitchell (1961). ATP is synthesized
when reconstituted membrane vesicles
containing bacteriorhodopsin (a light-driven
proton pump) and ATP synthase are
illuminated. The orientation of ATP synthase in
this reconstituted membrane is the reverse of
that in the mitochondrion.
The structure of F0 F1 ATPase

F0, 10-14 c subunits, Proton conduit.

F1, (α3β3γδε) contains the ATP synthase activity

(even in isolation)
Connectors, i> γε stalk in the inside.
ii> Two b subunits connect a and δ on
the outside.

we can think of the enzyme as consisting of two

functional components: (1) a moving unit, or rotor,
consisting of the c ring and the γε stalk, and (2) a
stationary unit, or stator, composed of the remainder
of the molecule.
No. 161
 Rotation of the c-complex

Proton Motion Across the Membrane Drives Rotation of the C Ring. A proton enters from
the intermembrane space into the cytosolic half-channel to neutralize the charge on an
aspartate residue in a c subunit. With this charge neutralized, the c ring can rotate
clockwise by one c subunit, moving an aspartic acid residue out of the membrane into the
matrix half-channel. This proton can move into the matrix, resetting the system to its initial
state.
 Thus, the difference in proton
concentration and potential on
the two sides of the membrane
leads to different probabilities of
protonation through the two half-
channels, which yields
directional rotational motion.
Each proton moves through the
membrane by riding around on
the rotating c ring to exit through
the matrix half-channel

Each 360-degree rotation of the γ

subunit leads to the synthesis and
release of three molecules of ATP.
Proton Path Through the Membrane. Each proton
enters the cytosolic half-channel, follows a
complete rotation of the c ring, and exits through
the other half-channel into the matrix.
 Paying the cytosolic NADH debt

Glycerol 3-Phosphate Shuttle. Electrons from NADH can enter the mitochondrial electron
transport chain by being used to reduce dihydroxyacetone phosphate to glycerol 3-
phosphate. Glycerol 3-phosphate is reoxidized by electron transfer to an FAD prosthetic
group in a membrane-bound glycerol 3-phosphate dehydrogenase. Subsequent electron
transfer to Q to form QH2 allows these electrons to enter the electron-transport chain.
 Paying the cytosolic NADH debt

When cytosolic NADH transported by the

glycerol 3-phosphate shuttle is oxidized by the
respiratory chain, 1.5 rather than 2.5 ATP are
formed. The yield is lower because FAD rather
than NAD+ is the electron acceptor in
mitochondrial glycerol 3-phosphate
dehydrogenase. The use of FAD enables
electrons from cytosolic NADH to be
transported into mitochondria against an NADH
concentration gradient. The price of this
transport is one molecule of ATP per two
electrons.

This glycerol 3-phosphate shuttle is especially prominent in muscle and

enables it to sustain a very high rate of oxidative phosphorylation. Indeed,
some insects lack lactate dehydrogenase and are completely dependent on the
glycerol 3-phosphate shuttle for the regeneration of cytosolic NAD +.
 Paying the cytosolic NADH debt

Malate-Aspartate Shuttle. In the heart and liver, electrons from cytosolic NADH are brought into mitochondria
by the malate-aspartate shuttle, which is mediated by two membrane carriers and four enzymes. Electrons
are transferred from NADH in the cytosol to oxaloacetate, forming malate, which traverses the inner
mitochondrial membrane and is then reoxidized by NAD + in the matrix to form NADH in a reaction catalyzed
by the citric acid cycle enzyme malate dehydrogenase. The resulting oxaloacetate does not readily cross
the inner mitochondrial membrane, and so a transamination reaction is needed to form aspartate, which can
be transported to the cytosolic side. Mitochondrial glutamate donates an amino group, forming aspartate and
α-ketoglutarate. In the cytoplasm, aspartate is then deaminated to form oxaloacetate and the cycle is
restarted. This shuttle, in contrast with the glycerol 3-phosphate shuttle, is readily reversible.
Consequently, NADH can be brought into mitochondria by the malate- aspartate shuttle only if the
NADH/NAD+ ratio is higher in the cytosol than in the mitochondrial matrix. This versatile shuttle also
facilitates the exchange of key intermediates between mitochondria and the cytosol.
The Entry of ADP into Mitochondria Is Coupled to the Exit
of ATP by ATP-ADP Translocase

Mechanism of Mitochondrial ATP-ADP Translocase. The translocase catalyzes the coupled

entry of ADP and exit of ATP into and from the matrix. The reaction cycle is driven by
membrane potential. The actual conformational change corresponding to eversion of the
binding site could be quite small.
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, cytosolic 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
 Mitochondrial carriers

Structure of Mitochondrial
Transporters. Many mitochondrial
transporters consist of three similar
100-residue units. These proteins
Mitochondrial Transporters. Transporters (also called carriers)
contain six putative membrane-
are transmembrane proteins that move ions and charged spanning segments.
metabolites across the inner mitochondrial membrane.

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 Pi 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 P i. 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 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
Electron transport chain
Electron transport chain
The best current estimates for the
number of protons pumped out of the
matrix by NADH-Q oxidoreductase, Q-
cytochrome c oxidoreductase, and
cytochrome c oxidase per electron pair
are four, two, and four, respectively.
The synthesis of a molecule of ATP is
driven by the flow of about three protons
through ATP synthase. An additional
proton is consumed in transporting
ATP from the matrix to the cytosol.
Hence, about 2.5 molecules of cytosolic
ATP are generated as a result of the flow
of a pair of electrons from NADH to O2.

For electrons that enter at the level of Q-

cytochrome c oxidoreductase, such as those
from the oxidation of succinate or cytosolic
NADH, the yield is about 1.5 molecules of ATP
per electron pair. Hence about 30 molecules of
ATP are formed when glucose is completely
oxidized to CO2; this value supersedes the
traditional estimate of 36 molecules of ATP.
The rate of oxidative phosphorylation is determined by the need of ATP

Electrons do not usually flow through the electron-transport chain to O 2unless ADP is
simultaneously phosphorylated to ATP. Oxidative phosphorylation requires a supply of NADH (or
other source of electrons at high potential), O2, ADP, and Pi. The most important factor in
determining the rate of oxidative phosphorylation is the level of ADP. The rate of oxygen
consumption by mitochondria increases markedly when ADP is added and then returns to its initial
value when the added ADP has been converted into ATP.
The regulation of the rate of oxidative phosphorylation by the ADP level is called respiratory control
or acceptor control.
Electron transport chain and oxidative phosphorylation
is inhibited by many chemicals

• Different inhibitors inhibit at different levels.

• Block of ATP synthase…Oligomycin and

Dicyclohexylcarbodimide
• Block of ATP-synthase causes Electron transport
chain to cease.
• Uncouplers e.g. 2,4-dinitrophenol and certain other
acidic aromatic compounds carry
protons across the inner mitochon-
-drial membrane.
• ATP-ADP translocase is specifically inhibited by very
low concentrations of atractyloside (a plant glycoside)
or bongkrekic acid (an antibiotic from a mold).
Atractyloside binds to the translocase when its
nucleotide site faces the cytosol, whereas bongkrekic
acid binds when this site faces the mitochondrial
matrix. Oxidative phosphorylation stops soon after
either inhibitor is added, showing that ATP-ADP
translocase is essential.
 Regulated Uncoupling Leads to the Generation of Heat
Brown fat mitochondria

Action of an Uncoupling Protein. Uncoupling protein-1 (UCP-1) generates heat by

permitting the influx of protons into the mitochondria without the synthesis of ATP.
 Cool facts

In addition to UCP-1, two other uncoupling proteins have been identified. UCP-2,
which is 56% identical in sequence with UCP-1, is found in a wide variety of
tissues. UCP-3 (57% identical with UCP-1 and 73% identical with UCP-2) is
localized to skeletal muscle and brown fat. This family of uncoupling proteins,
especially UCP-2 and UCP-3, may play a role in energy homeostasis. In fact, the
genes for UCP-2 and UCP-3 map to regions of the human and mouse
chromosomes that have been linked to obesity, substantiating the notion that
they function as a means of regulating body weight. The use of uncoupling
proteins is not limited to animals, however. The skunk cabbage uses an
analogous mechanism to heat its floral spikes, increasing the evaporation of
odoriferous molecules that attract insects to fertilize its flowers.
 Hazards of electron transport chain
Molecular oxygen is an ideal terminal electron acceptor, because its high
affinity for electrons provides a large thermodynamic driving force. However,
danger lurks in the reduction of O2. The transfer of four electrons leads to safe

products (two molecules of H2O), but partial reduction generates hazardous

compounds. In particular, the transfer of a single electron to O2forms

superoxide anion, whereas the transfer of two electrons yields peroxide.

These compounds and, particularly, their reaction products can be quite harmful to a
variety of cell components. The strategy for the safe reduction of O 2 is clear from the
discussion of the reaction cycle: the catalyst does not release partly reduced
intermediates. Cytochrome c oxidase meets this crucial criterion by holding O2 tightly
between Fe and Cu ions.
 The fix

The hydrogen peroxide formed by

superoxide dismutase and by other
processes is scavenged by catalase, a
ubiquitous heme protein that catalyzes the
dismutation of hydrogen peroxide into
water and molecular oxygen.

Superoxide dismutase and catalase are

Superoxide Dismutase Mechanism. The oxidized form
of superoxide dismutase (Mox) reacts with one superoxide
ion to form O2 and generate the reduced form of the
enzyme (Mred). The reduced form then reacts with a
second superoxide and two protons to form hydrogen
peroxide and regenerate the oxidized form of the enzyme.
remarkably efficient, performing their
reactions at or near the diffusion-limited
rate. Other cellular defenses against
oxidative damage include the antioxidant
vitamins, vitamins E and C. Because it is
lipophilic, vitamin E is especially useful in
protecting membranes from lipid
peroxidation.