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Consortium for
Educational
Communication
Module on
OXIDATIVE
PHOSPHORYLATION
AND SUBSTRATE-LEVEL
PHOSPHORYLATION
By
Parvaiz Ahmad
Assistant Professor
Govt. Degree College Anantnag
[email protected]
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Plants, algae and some bacteria harvest the energy of
sunlight through photosynthesis and convert radiant energy
into chemical energy. The reduced cellular carbon generated
during photosynthesis is oxidized to CO2 and water, and this
oxidation is coupled to the synthesis of ATP. Respiration takes
place in three main stages: glycolysis, the citric acid cycle and
oxidative phosphorylation. The latter comprises the electron
transport chain and ATP synthesis.
During glycolysis, carbohydrate is converted to pyruvate
in the cytosol, and a small amount of ATP is synthesized via
substrate-level phosphorylation. Pyruvate is subsequently
oxidized within the mitochondrial matrix through the citric acid
cycle, generating a large amount of reducing equivalents in the
form of NADH and FADH2.
In the third stage of oxidative phosphorylation, electrons
from NADH and FADH2 pass through the electron transport chain
in the inner mitochondrial membrane to reduce oxygen. The
chemical energy is conserved in the form of an electrochemical
proton gradient, which is created by the coupling of electron
flow to pumping of proton from the matrix to the intermembrane
space. This energy is then converted into chemical energy in
the form of ATP by the F0F1-ATP synthase, also located in the
inner membrane, that couples ATP synthesis (from ADP and
Pi) to the flow of protons back in to the matrix down their
electrochemical gradient.
Phosphorylation
Phosphorylation is the addition of a phosphate (PO43) group to glucose or other organic molecules like proteins
or fats to obtain energy in the form of ATP. ATP, the “highenergy” exchange medium in the cell, is synthesized in the
mitochondrion by addition of a third phosphate group to ADP
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in a process referred to as oxidative phosphorylation. ATP is
also synthesized by substrate-level phosphorylation during
glycolysis. ATP is synthesized at the expense of solar energy by
photophosphorylation in the chloroplasts of plant cells.
Oxidative phosphorylation
Oxidative phosphorylation is a process in which ATP is
formed as a result of transfer of electrons from NADH to FADH2
by a series of electron carriers. The electron transport chain
generates no ATP directly. Only 4 of 38 ATP ultimately produced
during respiration of glucose are derived from substrate-level
phosphorylation (2 from glycolysis and 2 from TCA cycle). The
vast majority of the ATP (90%) comes from the energy in the
electrons carried by NADH and FADH2.
During oxidative phosphorylation, electrons are transferred
from electron donors to electron acceptors such as oxygen, in
redox reactions. These redox reactions release energy, which
is used to form ATP. These redox reactions are carried out by a
series of protein complexes within the cell membrane in both
prokaryotes and eukaryotes. These proteins are located in the
cells intermembrane space. These linked sets of proteins form
the electron transport chains. In eukaryotes, five main protein
complexes are involved, whereas in prokaryotes many different
enzymes are present, using a variety of electron donors and
acceptors.
The energy released by electrons flowing through this
electron transport chain is used to transport protons across the
inner mitochondrial membrane, in a process called chemiosmosis.
This generates potential energy in the form of a pH gradient
and an electrical potential across this membrane. This store of
energy is tapped by allowing protons to flow back across the
membrane and down this gradient, through a large enzyme
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called ATP synthase. This enzyme uses this energy to generate
ATP from adenosine diphosphate (ADP), in a phosphorylation
reaction. This reaction is driven by the proton flow, which forces
the rotation of a part of the enzyme; the ATP synthase is a
rotary mechanical motor.
Although oxidative phosphorylation is a vital part of
metabolism, it produces reactive oxygen species such as
superoxide and hydrogen peroxide, which lead to propagation
of free radicals, damaging cells and contributing to disease
and possibly aging (senescence). The enzymes carrying out
this metabolic pathway are also the target of many drugs and
poisons that inhibit their activities.
Oxidative phosphorylation works by using energy-releasing
chemical reactions to drive energy-requiring reactions. The two
sets of reactions are said to be coupled. This means one cannot
occur without the other. The flow of electrons through the electron
transport chain, from electron donors such as NADH to electron
acceptors such as oxygen, is an exergonic process–it releases
energy, whereas the synthesis of ATP is an endergonic process,
which requires an input of energy. Both the electron transport
chain and the ATP synthase are embedded in a membrane, and
energy is transferred from electron transport chain to the ATP
synthase by movements of protons across this membrane, in
a process called chemiosmosis. In practice, this is like a simple
electric circuit, with a current of protons being driven from
the negative N-side of the membrane to the positive P-side by
the proton-pumping enzymes of the electron transport chain.
These enzymes are like a battery, as they perform work to drive
current through the circuit. The movement of protons creates
an electrochemical gradient across the membrane, which is
often called the proton-motive force. It has two components:
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a difference in proton concentration (a H+ gradient, ΔpH) and a
difference in electric potential, with the N-side having a negative
charge.
ATP synthase releases this stored energy by completing the
circuit and allowing protons to flow down the electrochemical
gradient, back to the N-side of the membrane. This kinetic energy
drives the rotation of part of the enzymes structure and couples
this motion to the synthesis of ATP.
The amount of energy released by oxidative phosphorylation
is high, compared with the amount produced by anaerobic
fermentation. Glycolysis produces only 2 ATP molecules, but
somewhere between 30 and 36 ATPs are produced by the oxidative
phosphorylation of the 10 NADH and 2 succinate molecules
made by converting one molecule of glucose to carbon dioxide
and water, while each cycle of beta oxidation of a fatty acid
yields about 14 ATPs. These ATP yields are theoretical maximum
values; in practice, some protons leak across the membrane,
lowering the yield of ATP.
History of Oxidative Phosphorylation
The field of oxidative phosphorylation began with the report
in 1906 by Arthur Harden of a vital role for phosphate in cellular
fermentation, but initially only sugar phosphates were known
to be involved. However, in the early 1940s, the link between
the oxidation of sugars and the generation of ATP was firmly
established by Herman Kalckar, confirming the central role of
ATP in energy transfer that had been proposed by Fritz Albert
Lipmann in 1941. Later, in 1949, Morris Friedkin and Albert L.
Lehninger proved that the coenzyme NADH linked metabolic
pathways such as the citric acid cycle and the synthesis of ATP.
For another twenty years, the mechanism by which ATP is
generated remained mysterious, with scientists searching for
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an elusive “high-energy intermediate” that would link oxidation
and phosphorylation reactions. This puzzle was solved by Peter
D. Mitchell with the publication of the chemiosmotic theory in
1961. At first, this proposal was highly controversial, but it
was slowly accepted and Mitchell was awarded a Nobel prize
in 1978. Subsequent research concentrated on purifying and
characterizing the enzymes involved, with major contributions
being made by David E. Green on the complexes of the electrontransport chain, as well as Efraim Racker on the ATP synthase. A
critical step towards solving the mechanism of the ATP synthase
was provided by Paul D. Boyer, by his development in 1973
of the “binding change” mechanism, followed by his radical
proposal of rotational catalysis in 1982. More recent work has
included structural studies on the enzymes involved in oxidative
phosphorylation by John E. Walker, with Walker and Boyer being
awarded a Nobel Prize in 1997.
Mechanism of Oxidative Phosphorylation
Three important theories have been proposed to explain the
mechanism of oxidative phosphorylation. These theories explain
how the energy transfer between electron transport and ATP
synthesis takes place. These are chemical coupling hypothesis,
conformational coupling hypothesis and the chemiosmotic
hypothesis.
Chemical Coupling Hypothesis
It was first proposed by Slater in 1953 and is based on
the principles of substrate level phosphorylation. The hypothesis
postulates that a high energy intermediate is produced as
electrons are passed from one carrier to the next. However, no
such high energy intermediates have been shown to exist and the
need for intact mitochondrial membranes for effective oxidative
phosphorylation is not explained by this hypothesis.
For the explanation of hypothesis, it is proposed that two
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hypothetical coupling factors (enzymes), called X and E, are
involved at each ATP generating step. It is further proposed
that coupling factors required at three ATP generating steps are
different. They are designated as X1 and E1, X2 and E2 and X3 and
E3. The various steps of mechanism are as follows:
(i) The coupling factor X first combines with the respiratory
enzyme like Cyt. b to form a high energy intermediate
complex (Fe3+~ X).
(ii) The high energy intermediate complex combines with PO4to form phosphorylated intermediate (X~P) containing a
high energy phosphate group. At this step the respiratory
enzyme is removed.
(iii) The phosphorylated intermediate (X~P) now combines
with another coupling factor E to replace first coupling
factor X and to form energy rich phosphorylated complex
(E~P) which catalyses the synthesis of ATP from ADP and
regeneration of the coupling factor E.
Thus, it is presumed that the function of coupling factors X
and E is to transfer the energy released in redox reactions for
ATP synthesis.
Conformational Coupling Hypothesis
It was first proposed by Boyer in 1964. According to this
hypothesis, the energy produced during electron transfer
is converted by conformational changes in the molecules
comprising the mitochondrial membrane and matrix which may
be the driving force for ATP formation. The main conformational
changes have been observed in ATP synthetase particles and
cristae of mitochondria. The cristae assume different forms
during different functional states of the mitochondrion. Similarly,
the ATP synthetase particles also assume different shapes like
disc, dumb-bell or spherical. When there is lack of energy supply
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and the mitochondrion is in non-energized state, the cristae are
in the form of straight flattened sacs and stalk of ATP synthetase
particles, also called repeating units, becomes contracted and
its head piece or F1 part becomes flattened to form a disc.
When isolated mitochondria are supplied the substrates or kept
in solution containing ATP, they are converted in to energized
state. At this stage, the cristae become more organized and
assume vesicular form whereas the stems of ATP synthetase
particles assume an extended form and F1 part becomes dumbbell shaped. When only inorganic phosphate and no ADP is
supplied to mitochondria, the membranes of the cristae become
convoluted assuming zig-zag shape (energy twisted) whereas
the stem of ATP synthetase particles becomes elongated and F1
part becomes spherical. When ADP is added, the energy-twisted
state is changed to energized state which can be converted to
non-energized state by addition of such cations or uncouplers
that can be transported across the membrane.
Of the above conformational changes, the twisted form
stores the energy released during electron transport. When
energy is added for ATP synthesis, the energy twisted or
energized state returns to stable non-energized state releasing
energy which is utilized for the synthesis of ATP from ADP and
inorganic phosphate. Boyer (1965) proposed that there is a
direct communication between electron transfer catalysts and
ATP synthesizing components through polypeptide polypeptide
interaction.
Boyer and Slater (1974) proposed a modified conformational
coupling hypothesis which postulates that electron transfer induces
conformational changes leading to translocation of protons.
Conformational changes in electron transfer proteins induce
changes in ATP synthesizing protein components. They believe
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that passage of protons through F1 can change the conformation
of its protein and such proton induced conformational changes
near the active site can synthesize ATP.
ADP and inorganic phosphate can combine spontaneously
to form ATP in the active site of F1 of ATPase without requiring
free energy. ATP formed is tightly bound to ATPase. The energy
is, therefore, required to release tightly bound ATP molecules
from ATPase. The protons when bind elsewhere other than the
active site, can cause conformational changes in F1 part of ATPase
resulting in to release of ATP. The protons are released in to the
solution on M-side of the membrane.
The Chemiosmotic Hypothesis
It was proposed by a British biochemist, Peter Mitchell, in
1961. This theory is most convincing and acceptable to date.
According to the chemiosmotic theory, the orientation of electron
carriers within the mitochondrial inner membrane allows for
the transfer of protons (H+) across the inner membrane during
electron flow. Because the inner mitochondrial membrane is
impermeable to H+, an electrochemical proton gradient can build
up.
As the proton concentration in the intermembrane space
rises above that in the matrix, the matrix becomes slightly
negative in charge. This internal negativity attracts the positively
charges protons and induces them to reenter the matrix. The
higher outer concentration tends to drive protons back in by
diffusion; since membranes are relatively impermeable to ions,
most of the protons that reenter the matrix pass through special
proton channels in the inner mirtochondrial membrane. When
the protons pass through the, these channels synthesize ATP
from ADP + Pi within the matrix. The ATP is then transported
by facilitated diffusion out of the mitochondrion and in to the
cell’s cytoplasm. Because the chemical formation of ATP is
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driven by a diffusion force similar to osmosis, this process is
referred to as chemiosmosis. Thus, the electron transport chain
uses electrons harvested in aerobic respiration to pump a large
number of protons across the inner mitochondrial membrane.
Their subsequent reentry in to the mitochondrial matrix drives
the synthesis of ATP by chemiosmosis.
The chemiosmotic model suggests that one ATP molecule
is generated for each proton pump activated by the electron
transport chain. As the electron from NADH activate three
pumps and those from FADH2 activate two, thus each molecule of
NADH and FADH2 would generate three and two ATP molecules,
respectively. However, because eukaryotic cells carry out
glycolysis in their cytoplasm and their Krebs cycle within their
mitochondria, they must transport the two molecules of NADH
produced during glycolysis across the mitochondrial membranes,
which requires one ATP molecule per molecule of NADH. Thus, the
net ATP production is decreased by two. Therefore, the overall ATP
production resulting from aerobic respiration theoretically should
be 4 (from substrate-level phosphorylation during glycolysis) +
30 (3 from each of 10 molecules of NADH) + 4 (2 from each of
two molecules of FADH2) – 2 (for transport of glycolytic NADH)
= 36 molecules of ATP. But the amount of ATP produced in a
eukaryotic cell during aerobic respiration is somewhat lower than
36, for two reasons. First, the inner mitochondrial membrane is
somewhat leaky to protons, allowing some of them to reenter
the matrix without passing through ATP-generating channels.
Second, mitochondria often use the proton gradient generated
by chemiosmosis for purposes other than ATP synthesis (such
as transporting pyruvate in to the matrix). Consequently, the
actual measured values of ATP generated by NADH and FADH2
are closer to 2.5 for each NADH and 1.5 for each FADH2. With
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these corrections, the overall harvest of ATP from a molecule of
glucose in a eukaryotic cell is closer to 4 (from substrate-level
phosphorylation) + 25 (2.5 from each of 10 molecules of NADH)
+ 3 (from each of two molecules of FADH2) – 2 (transport of
glycolytic NADH) = 30 molecules of ATP.
Evidences in support of Chemiosmotic Hypothesis
There are a number of evidences in the support of
chemiosmotic hypothesis for oxidative phosphorylation but
the most important one came by the use of chemicals like
2,4-dinitrophenol, during phosphorylation studies. These
chemicals destroyed the proton gradient across mitochondrial
membranes preventing ATP synthesis and were called as
uncouplers. ATP was further synthesized when pH (proton)
gradient was imposed on mitochondria in absence of electron
transport.
The inner mitochondrial membrane is impermeable to H+,
K+, OH-, and Cl-. If the membrane is damaged in order to pass
through such ions readily, oxidative phosphorylation will not
take place. If the vectorial organization of respiratory chain
and ATPase in the coupling membrane is changed, oxidative
phosphorylation does not take place.
Inhibitors of electron transfer
There are several well-known drugs and toxins that inhibit
oxidative phosphorylation. Although any one of these toxins
inhibits only one enzyme in the electron transport chain, inhibition
of any step in this process halts the rest of the process. Three
inhibitors have been found to block electron transport between
NADH and ubiquinone:
(i) Rotenone, an extremely toxic plant substance.
(ii) Amytal, a barbiturate.
(iii)Piercidin, an antibiotic that resembles ubiquinone in
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structure.
These compounds are believed to act on NADH dehydrogenase.
Another characteristic inhibitor is the antibiotic antimycin
A, isolated from Streptomyces griseus which block electron
transport in the span of cytochrome b to c. A third class of
inhibitor blocks electron transport from cytochrome a-a3 to
oxygen; it includes hydrogen cyanide, hydrogen sulphode, and
carbon monoxide. The site of action of these inhibitors has been
established by spectrophotometric measurement of the oxidation
reduction states of the different electron carriers before and after
application of the inhibitor to actively respiring mitochondria,
which produces a crossover point.
Uncouplers
Uncouplers are the compounds that increase the permeability
of the inner mitochondrial membrane to protons. Protons enter
the matrix at sites other than ATP synthase through holes made by
these compounds. These compounds have no effect on electron
transport chain, but they uncouple oxidation phosphorylation.
The energy produced by the transport of electrons is released
as heat rather than by being used to synthesize ATP. Some
examples of uncouplers are 2,4-dinitrophenol, dinitrocresol,
Pentachlorophenol, Thyroxine, Calcium etc.
Substrate-level phosphorylation
Substrate-level phosphorylation is a type of metabolism that
results in the formation and creation of adenosine triphosphate
(ATP) or guanosine triphosphate (GTP) by the direct transfer
and donation of a phosphoryl group to adenosine diphosphate
(ADP) or guanosine diphosphate (GDP) from a phosphorylated
reactive intermediate. The phosphoryl group that is transferred
is referred to as a phosphate group.
Unlike
oxidative
phosphorylation,
oxidation
and
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phosphorylation are not joined in the process of substrate-level
phosphorylation, although both types of phosphorylation result
in ATP and reactive intermediates are most often gained in course
of oxidation processes in catabolism. However, usually most of
the ATP is generated by oxidative phosphorylation in aerobic or
anaerobic respiration. Substrate-level phosphorylation serves
as fast source of ATP independent of external electron acceptors
and respiration. The main part of Substrate-level phosphorylation
occurs in the cytoplasm of cells as part of glycolysis and in
mitochondria as part of the Krebs Cycle under both aerobic and
anaerobic conditions.
In the pay-off phase of glycolysis, four ATP are produced
by substrate-level phosphorylation: two and only two
1,3-bisphosphoglycerate are converted to 3-phosphoglycerate
by transferring a phosphate group to ADP by a kinase; two
phosphoenolpyruvate are converted to pyruvate by the transfer
of their phosphate groups to ADP by another kinase. The first
reaction occurs after the generation of 1,3-bisphosphoglycerate
from 3-phosphoglyceraldehyde and an organic phosphate via a
dehydrogenase. ATP is generated by transfer of the high-energy
phosphate on 1,3-bisphosphoglycerate to ADP via the enzyme
phosphoglycerate kinase, generating 3-phosphoglycerate. As
ATP is formed of a former inorganic phosphate group, this step
leads to the energy yield of glycolysis. The second Substratelevel phosphorylation occurs later by means of the reaction
of phosphenolpyruvate (PEP) to pyruvate via the pyruvate
kinase. This reaction regenerates the ATP that has been used
in the preparatory phase of glycolysis to activate glucose to
glucose-6-phosphate and fructose-6-phosphate to fructose-1,6bisphosphate, respectively.
Once the pyruvate product of glycolysis is moved to the
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mitochondrial matrix, the pyruvate is converted to acetate
and binds Coenzyme A to form Acetyl CoA to enter the Krebs
cycle. While the Krebs cycle is oxidative respiration, one more
instance of substrate-level phosphorylation occurs as Guanosine
triphosphate (GTP) is created from GDP by transfer of a phosphate
group during the conversion of Succinyl CoA to Succinate. This
phosphate is transferred to ADP in another substrate-level
phosphorylation event. The reaction is catalyzed by the enzyme
Succinyl-CoA synthetase.