UNIT - II

BIOCHEMISTRY & METABOLISM

CARBOHYDRATE METABOLISM
 Glycolysis
Glycolysis: Derived from Greek words;

Glykys = Sweet, Lysis = splitting

During this process one molecule of glucose (6 carbon molecule)
is degraded into two molecules of pyruvate (three carbon
molecule).

Free energy released in this process is stored as 2 molecules of

ATP, and 2 molecules of NADH.

δGo (overall) = -146+61 = -85 kJ/mol

In standard condition glycolysis is an exergonic reaction

 Fate of glucose in living systems

Glucose + 6O2 = 6CO2 + 6H2O δGo= -2840 kJ/mol

Glucose + 2NAD+ = 2Pyruvate + 2NADH + 2H+ δGo = -146 kJ/mol

5.2% of total free energy that can be released by glucose is released in

glycolysis.
There are 10 enzyme-catalyzed reactions in
glycolysis. There are two stages
Stage 1: Reactions 1-5) A preparatory stage in which glucose
is phosphorylated, converted to fructose which is again
phosphorylated and cleaved into two molecules of
glyceraldehyde-3-phosphate. In this phase there is an
investment of two molecules of ATP.
Stage 2: (reactions 6-10) The two molecules of
glyceraldehyde-3-phosphate are converted to pyruvate with
concomitant generation of four ATP molecules and two
molecules of NADH. Thus there is a net gain of two ATP
molecules per molecule of Glucose in glycolysis.
1. Hexokinase reaction: phosphorylation of hexoses (mainly
glucose)

I. This enzyme is present in most cells. In liver Glucokinase is the

main hexokinase which prefers glucose as substrate.
II. It requires Mg-ATP complex as substrate. Un-complexed ATP is a
potent competitive inhibitor of this enzyme.
III. Enzyme catalyses the reaction by proximity effect; bringing the two
substrate in close proximity.
IV. This enzyme undergoes large conformational change upon binding
with Glucose. It is inhibited allosterically by G6P.
2. Phosphoglucose Isomerase or Phosphohexose
Isomerase: Isomerization of G6P to Fructose 6
phosphate.

I. This enzyme catalyzes the reversible isomerization of G6P (an

aldohexose) to F6P (a ketohexose).
II. This enzyme requires Mg ++ for its activity.
III. It is specific for G6P and F6P.
3. Phosphofructokinase-1 Reaction: Transfer of
phosphoryl group from ATP to C-1 of
F6P to produce Fructose 1,6 biphosphate.

I. This step is an important irreversible, regulatory step.

II. The enzyme Phosphofructokinase-1 is one of the most complex
regulatory enzymes, with various allosteric inhibitors and
activators.
III. ATP is an allosteric inhibitor, and Fructose 2,6 biphosphate
is an activator of this enzyme.
IV. ADP and AMP also activate PFK-1 whereas citrate is an inhibitor.
4. Aldolase Reaction: Cleavage of Fructose 1,6
biphosphate into glyceraldehyde 3 phosphate (an
aldose) and dihydroxy acetone phosphate (a ketose).

I. This enzyme catalyses the cleavage of F1,6 biphosphate by aldol

condensation mechanism.
II. As shown below, the standard free energy change is positive in the
forward direction, meaning it requires energy. Since the product of
this reaction are depleted very fast in the cells, this reaction is
driven in forward direction by the later two reactions.
5. Triose phosphate mutase reaction: Conversion of
Dihydroxyacetone phosphate to glyceraldehyde 3
Phosphate.

I. This a reversible reaction catalysed by acid-base catalysis in which

Histidine-95 and Glutamate -165 of the enzyme are involved.
6. Glyceraldehyde-3-phosphate dehydrogenase reaction (GAPDH):
Conversion of GAP to Bisphosphoglycerate.

I. This is the first reaction of energy yielding step. Oxidation of

aldehyde derives the formation of a high energy acyl phosphate
derivative.
II. An inorganic phosphate is incorporated in this reaction without any
expense of ATP.
III. NAD+ is the cofactor in this reaction which acts as an oxidizing
agent. The free energy released in the oxidation reaction is used in
the formation of acylphosphate.
7. Phosphoglycerate kinase Reaction: Transfer of phosphoryl
group fron 1,3 bisphosphoglycerate to ADP generating ATP.
I. The name of this enzyme indicates its function for reverse
reaction.
II. It catalyses the formation by proximity effect. ADP-Mg bind on
one domain and 1,3BPG binds on the other and a conformational
change brings them together similar to hexokinase.
III. This step generates ATP by SUBSTRATE-LEVEL
PHOSPHORYLATION.
8. Phosphoglycerate Mutase Reaction: Conversion of 3-
phosphoglycerate to 2-phosphoglycerate (2-PG).
I. In active form, the phosphoglycerate mutase is phosphorylated at
His-179.
II. There is transfer of the phosphoryl group from enzyme to 3-PG,
generating enzyme bound 2,3-biphosphoglycerate intermediate.
This compound has been observed occasionally in reaction
mixture.
III. In the last step of reaction the phosphoryl group from the C-3 of
the intermediate is transferred to the enzyme and 2-PG is
released.
IV. In most cells 2,3BPG is present in trace amount, but in
erythrocytes it is present in significant amount. There it regulates
oxygen affinity to hemoglobin.
9. Enolase Reaction: Dehydration of 2-phosphoglycerate
(2-PG) to phosphoenolpyruvate (PEP).

I. Dehydration of 2-PG by this reaction increases the standard free

energy change of hydrolysis of phosphoanhydride bond.
II. Mechanism: Rapid extraction of proton from C-2 position by a
general base on enzyme, generating a carbanion.
III. The second rate limiting step involves elimination of OH group
generating PEP
10. Pyruvate Kinase Reaction: Transfer of phosphoryl group
from PEP to ADP generating ATP and Pyruvate.

I. This is the second substrate level phosphorylation reaction of

glycolysis.
II. This enzyme couple the free energy of PEP hydrolysis to the
synthesis of ATP
III. This enzyme requires Mg++ and K+
Regulation of Glycolysis:

Two types controls for metabolic reactions:

a) Substrate limited : When concentrations of reactant and products in

the cell are near equilibrium, then it is the availability of substrate
which decides the rate of reaction.

b) Enzyme-limited: When concentration of substrate and products are

far away from the equilibrium, then it is activity of enzyme that
decides the rate of reaction. These reactions are the one which
control the flux of the overall pathway.

There are three steps in glycolysis that have enzymes which regulate
the flux of glycolysis.

I. hexokinase (HK)
II. phoshofructokinase (PFK)
III. pyruvate kinase
TCA CYCLE
Citric Acid cycle or Tricarboxylic Acid cycle or Krebs Cycle
Overview and brief history

•Pyruvate Dehydrogenase Complex (PDC) and its control

•Reactions of TCA cycle or CAC

•Amphibolic nature of TCA cycle

•Regulation of TCA cycle

•Reactions of Glycolysis are localized in Cytosol, and do not require any

oxygen.

whereas pyruvate dehydrogenase and TCA cycle reactions take place in

mitochondria where oxygen is utilized to generate ATP by oxidative
phosphorylation.

Consumption of oxygen (respiration) depends on the rate of PDC and TCA

reactions.
 Respiration
• Cellular respiration occurs in three major stages
• I. organic fuel molecules—glucose,fatty acids, and some amino acids—
are oxidized to yield two-carbon fragments in the form of the acetyl
group of acetyl-coenzyme A (acetyl-CoA).
• II. In the second stage, the acetyl groups are fed into the citric acidcycle,
which enzymatically oxidizes them to CO2; the energy released is
conserved in the reduced electron carriers NADH and FADH2.
• III. In the third stage of respiration,these reduced coenzymes are
themselves oxidized, giving up protons (H) and electrons. The electrons
are transferred to O2—the final electron acceptor—via a chain of
electron-carrying molecules known as the respiratory chain.
• In the course of electron transfer, the large amount of energy released is
conserved in the form of ATP, by a process called oxidative
phosphorylation
In Cytosol

In Mitochondria
Reaction of pyruvate dehydrogenase complex (PDC)

Reactions of TCA cycle: 8 reactions:

Citrate synthase
Aconitase
Iso-citrate dehydrogenase
α ketoglutarate dehydrogenase
Succinyl-Coenzyme A synthetase
Succinate dehydrogenase
Fumerase
Malate dehydrogenase
Pyruvate dehydrogenase Complex (PDC)

It is a multi-enzyme complex containing three enzymes associated

together non-covalently:

E-1 : Pyruvate dehydrogenase , uses Thiamine pyrophosphate as

cofactor bound to E1

E-2 : Dihydrolipoyl transacetylase, Lipoic acid bound, CoA as

substrate

E-3 : Dihydrolipoyl Dehydrogenase FAD bound, NAD+ as substrate

Advantages of multienzyme complex:

1. Higher rate of reaction: Because product of one enzyme acts as a

substrate of other, and is available for the active site of next enzyme
without much diffusion.
2. Minimum side reaction.
3. Coordinated control.
» yellow - E1
» green - E2
» red - E3
» E1 catalyzes first the decarboxylation of pyruvate, producing
hydroxyethyl-TPP, and then the oxidation of the hydroxyethyl group
to an acetyl group.
» E2 catalyzes the transfer of the acetyl group to coenzyme A,
forming acetyl-CoA.
» E3 catalyzes the regeneration of the disulfide (oxidized) form of
lipoate; electrons pass first to FAD, then to NAD.
» The long lipoyllysine arm swings from the active site of E1 to E2 to
E3, tethering the intermediates to the enzyme complex to allow
substrate channeling.
Reactions of Citric Acid Cycle

1. Citrate synthase: Formation of Citroyl CoA intermediate.

Binding of Oxaloacetate to the enzyme results in conformational change
which facilitates the binding of the next substrate, the acetyl Coenzyme A.
There is a further conformational change which leads to formation of
products.
The reaction catalyzed by citrate synthase is essentially a Claisen
condensation involving a thioester (acetyl-CoA) and a ketone (oxaloacetate).
2. Aconitase: This enzyme catalyses the isomerization reaction by
removing and then adding back the water ( H and OH ) to cis-aconitate
at different positions. Isocitrate is consumed rapidly by the next step
thus driving the reaction in forward direction.
• Aconitase contains an ironsulfur center , which acts both in the binding
of the substrate at the active site and in the catalytic
addition or removal of H2O.
3. Isocitrate dehydrogenase: There are two isoforms of this enzyme, one
uses NAD+ and other uses NADP+ as electron acceptor.

• The main function of the NADP-dependent enzyme, found in both the

mitochondrial matrix and the cytosol, may be the generation of NADPH, which
is essential for reductive anabolic reactions.
4. α-Ketoglutarate dehydrogenase: This is a complex of different
enzymatic activities similar to the pyruvate dyhdogenase complex. It
has the same mechanism of reaction with E1, E2 and E3 enzyme units.
NAD+ is an electron acceptor.
• The energy of oxidation of -ketoglutarate is conserved in
the formation of the thioester bond of succinyl-CoA
• the E1 components of the two complexes are structurally
similar, their amino acid sequences differ and, of course,
they have different binding specificities:
• E1 of the PDH complex binds pyruvate, and E1 of the -
ketoglutarate dehydrogenase complex binds -
ketoglutarate.
• The E2 components of the two complexes are also very
similar, both having covalently bound lipoyl moieties.
• The subunits of E3 are identical in the two enzyme
complexes.
5. Succinyl CoA synthatase: Succinyl CoA, like Acetyl CoA has a
thioester bond. In this reaction, the hydrolysis of the thioester bond
leads to the formation of phosphoester bond with inorganic
phosphate. This phosphate is transferred to Histidine residue of the
enzyme and this high energy, unstable phosphate is finally transferred
to GDP resulting in the generation of GTP.
6. Succinate Dehydrogenase: Oxidation of succinate to fumarate. This is
the only citric acid cycle enzyme that is tightly bound to the inner
mitochondrial membrane. It is an FAD dependent enzyme.

Malonate has similar structure to Succinate, and it competitively inhibits

SDH.
7. Fumarase: Hydration of Fumarate to malate.
8. L-Malate dehydrogenase: Oxidation of malate to oxaloacetate: It is an
NAD+dependent enzyme. Reaction is pulled in forward direction by the
next reaction (citrate synthase reaction) as the oxaloacetate is depleted at
a very fast rate.
Conservation of energy of oxidation in the CAC: The two carbon acetyl
group generated in PDC reaction enter the CAC, and two molecules of CO2 are
released in one cycle. Thus there is complete oxidation of two carbons during
one cycle. Although the two carbons which enter the cycle become the part of
oxaloacetate, and are released as CO2 only in the third round of the cycle. The
energy released due to this oxidation is conserved in the reduction of 3 NAD+, 1
FAD molecule and synthesis of one GTP molecule which is converted to ATP.
Anaerobic bacteria use incomplete citric acid cycle for production of
biosynthetic precursors. They do not contain a-ketoglutarate
dehydrogenase.
Regulation of CAC:
Rate controlling enzymes:
Citrate synthase
Isocitrate dehydrogenase
α-ketoglutaratedehydrogenase

Regulation of activity by:

Substrate availability
Product inhibition
Allosteric inhibition or activation by
other intermediates
FATTY ACID METABOLISM
Fatty acid (FA) activation
before oxidation
All the enzymes involved in
oxidation of FA are present in
mitochondria. The free FA
obtained from blood cannot
enter mitochondia.
In the first step, FA are
converted to fatty acyl CoA on
the outer mitochodrial
membrane by an ezyme called
Fatty acyl CoA synthase (also
called thiokinase).
This reaction is coupled with
ATP hydrolysis to AMP, and 2Pi.
There are different isoforms of
Fatty acyl CoA synthase
specific for different kind of
FAs.
This is the regulatory step of FA
FA + ATP + CoA-SH = Fatty acyl-CoA + AMP + 2Pi
oxidation pathway.
δG = -34 KJ/mole
FA entry into mitochondria via Fatty acyl-carnitine-
carnitine transporter: Fatty acyl CoA ester formed on
outer mitochondrial membrane do not enter directly in
mitochondria. 1.The FA is transferred to OH gp of
carnitine by Carnityl acyl transferase I (CAT-I), then the
fatty acyl-Carnitine ester is transported in the
mitochondra. 2. In mitochondria, FA is transferred to
mitochondral CoA by CAT-II, and the Fatty acyl-CoA
thus formed is ready for oxidation pathway.
Cytosolic and mitochondrial CoA pools have different
functions; for biosynthetic and catabolic reaction
respectively.
Once in mitochondria, the fatty acyl
CoA is subjected to beta oxidation.

Utilization of FA for oxidation and

generation of ATP is achieved in the
following three steps;

1. beta-oxidation of fatty acid chain

yielding acaty-CoA.

2. Entry of actyl-CoA in citric acid

cycle yielding NADH, FADH2 and GTP.

3. Utilization of NADH and FADH2 in

oxidative phosphorylation generating
ATP.

The first Fatty acyl-CoA

dehydrogenase enzyme of β-oxidation
pathway is linked to ETC and it directly
transfers the electrons to Coenzyme Q
in ETC via FADH2.
The β-Oxidation of fatty acyl-
CoA: 1. The first enzyme catalyses
the formation of a trans α, β double
bond, using FAD as cofactor. This
enzyme is linked to electron
transport chain via electron
transferring flavoprotein
2. Hydration of the double bond by
enoyl-CoA hydratase to form L-β-
hydroxyacyl-CoA.
3. NAD+-dependent
dehydrogenation by L-β-
hydroxyacyl-CoA dehydrogenase to
form β-ketoacyl-CoA.
4. Cα—Cβ cleavage in athiolysis
reaction with CoA, catalysed by
thiolase, producing acetyl-Coa and
a new fatty acyl-CoA with two less
carbon units.
The four steps of β-oxidation are
repeated to get FA completely
converted to acetyl-CoA.
For example for a 16 carbon fatty
acid, Palmityl-CoA, it will take 7
cycle of β-oxidation to generate 8
acetyl-CoA.
Thus there will be production of
7 FADH2, 7 NADH molecules
during the β-oxidation cycles.

From 8 acetyl-CoA there will be

generation of;
8 GTPs, 8 FADH2, 24 NADH and
16 CO2
 Biosynthesis of Fatty Acids
The formation of malonyl-CoA from acetyl-CoA and bicarbonate is an irreversible process, catalyzed by
acetyl-CoA carboxylase.
 Addition of two carbons to a growing
fatty acyl chain: a four-step sequence.
» Each malonyl group and acetyl group is activated by a thioester that links
it to fatty acid synthase, a multienzyme complex
» 1 Condensation of an activated acyl group (an acetyl group from
acetyl-CoA is the first acyl group) and two carbons derived from
malonyl-CoA, with elimination of CO2 from the malonyl group,
extends the acyl chain by two carbons.
» 2 the -keto group is reduced to an alcohol
» 3 elimination of H2O creates a double bond
» 4 the double bond is reduced to form the corresponding saturated
fatty acyl group.
» fatty acid synthase system consists of seven
separate polypeptides
» Throughout the process, the intermediates
remain covalently attached as thioesters to one
of two thiol groups of the synthase complex.
» One point of attachment is the OSH group of a
Cys residue in one of the seven synthase
proteins ( -ketoacyl-ACP synthase); the other is
the OSH group of acyl carrier protein.
Biosynthesis of Fatty Acids
» Step 1 Condensation
» The first reaction in the formation of a fatty acid
chain is condensation of the activated acetyl
and malonyl groups to form acetoacetyl-ACP, an
acetoacetyl group bound to ACP through the
phosphopantetheine OSH group;
simultaneously, a molecule of CO2 is produced.
In this reaction, catalyzed by - ketoacyl-ACP
synthase (KS), the acetyl group is transferred
from the Cys OSH group of the enzyme to the
malonyl group on the OSH of ACP, becoming
the methyl-terminal two-carbon unit of the new
acetoacetyl group.
» Step 2
» Reduction of the Carbonyl Group The
acetoacetyl- ACP formed in the condensation step
now undergoes reduction of the carbonyl group at
C-3 to form D- - hydroxybutyryl-ACP. This reaction
is catalyzed by - ketoacyl-ACP reductase (KR)
and the electron donor is NADPH. Notice that the
D- -hydroxybutyryl group does not have the same
stereoisomeric form as the L- - hydroxyacyl
intermediate in fatty acid oxidation
» Step 3 Dehydration
» The elements of water are now re- moved from C-2 and C-3 of
D- -hydroxybutyryl-ACP to yield a double bond in the product,
trans- 2- butenoyl- ACP. The enzyme that catalyzes this
dehydration is - hydroxyacyl-ACP dehydratase (HD).
» Step 4 Reduction of the Double Bond
» Finally, the double bond of trans- 2-butenoyl-ACP is
reduced (saturated) to form butyryl-ACP by the action of
enoyl-ACP reductase (ER); again, NADPH is the electron
donor.
AMINO ACID CATABOLISM
 Overview of amino acid catabolism
in mammals
• Amino acids lose their amino groups to form 𝛂-keto acids, the
“carbon skeletons” of amino acids.
• The 𝛂-keto acids undergo oxidation to CO2 and H2O or, often
more importantly, provide three- and four-carbon units that can be
converted by gluconeogenesis into glucose, the fuel for brain,
skeletal muscle, and other tissues.
 Amino acid Catabolism
Amino acids:
1. There are 20 different amino acid, they are monomeric constituents of proteins
2. They act as precursors of other nitrogen containing biologically important
compounds, like hormones, neurotransmitters etc.
3. Can be used as energy source.

We will be discussing just the catabolism of the amino acids (AAs), to generate
energy.
There are three major steps in catabolism of AAs.
1. Removal of amino group: deamination by
I. Transamination : Transfer of amino gp to 𝛂-ketoglutarate yielding glutamate
II. Oxidative amination: removal of amino gp from glutamate to release
ammonia
III. Other deamination processes.

2. Urea Cycle: Conversion of NH3 to urea for excretion

3. Metabolic break down of carbon skeleton to generate common

intermediates that can be catabolized to CO2 or used in anabolic
pathways to be stored as glucose or fat.
Metabolic fates of Amino groups
• Most amino acids are metabolized in the liver.
• Some of the ammonia generated in this process is recycled and
used in a variety of biosynthetic pathways;
• the excess is either excreted directly or converted to urea or uric
acid for excretion, depending on the organism
Excretory forms of Nitrogen
Transamination: Transfer of amino group to 𝛂-ketoglutarate. There are several
aminotransferases specific to different amino acids. In this step amino group from all
the amino acids are transferred to 𝛂-ketoglutarate and they exist as glutamate.

Transaminases or aminotransferases require pyridoxal-5’-phophate PLP (vitamin B6

derivative)
PLP is very important cofactor for many enzymatic reactions.
Oxidative deamination: In liver the amino group of glutamate is
released as ammonia, regenerating 𝛂-ketoglutarate, by an enzyme glutamate
dehydrogenase.
Glutamate dehydrogenase requires NAD+ or NADP+ as cofactor. This is the
only enzyme known that has specificity for both type of cofactor.
This enzyme is allosterically inhibited by GTP and activated by ADP.
Transport of excess ammonia
by glutamine: Excess ammonia is
toxic to animal tissues. Other than
amino acid catabolism in tissues
ammonia is also produced as a result
of nucleic acid degradation.
Glutamine synthase catalyses the
synthesis of glutamine by adding the
ammonia to glutamate at the expense of
ATP hydrolysis.
Glutamine is a non-toxic carrier of
ammonia. It is transported to liver or
kidney via blood.
In liver or kidney mitochondria, the
glutamine is converted to glutamate
and ammonia. Ammonia is
incorporated in urea cycle in liver to be
excreted.
Glucose-Alanine cycle:
Amino group from excess
glutamate produced in muscle as a
result of amino acid catabolism, is
transferred to pyruvate resulting in
the formation of alanine.
Alanine is another safe way to
transport ammonia from muscle to
liver via blood.
In liver alanine aminotransferase
transfers the amino gp to glutarate
and pyruvate regenerated is used
in gluconeogenesis.
Glucose produced by
gluconeogenesis is transported to
muscle where it enters the
glycolysis.
Thus the excess pyruvate and
ammonia generated in muscle are
safely transported to liver.
Carbamoyl phosphate
synthase-I Reaction:
Ammonia released from the
oxidative deamination is
incorporated in carbamoyl
phosphate by using ATP and
bicarbonate.

Carbamoyl phosphate enters

the urea cycle in the
mitochondria.
 Urea cycle
1. Ornithine transcarbomylase
2. Argininosuccinate synthetase
3. Argininosuccinase
4. Arginase
Interaction of Urea Cycle and Citric Acid Cycle via Aspartate-
Argininosuccinate shunt
OXIDATIVE PHOSPHORYLATION
» Oxygen needed so cells can use this molecule during
oxidative phosphorylation, the final stage of cellular
respiration.
» Oxidative phosphorylation is made up of two closely
connected components: the electron transport chain
and chemiosmosis.
» In ETC, electrons are passed from one molecule to
another, and energy released in these electron
transfers is used to form an electrochemical gradient.
» In chemiosmosis, the energy stored in the gradient
is used to make ATP.
» Oxygen sits at the end of the electron transport chain, where it
accepts electrons and picks up protons to form water.
» If oxygen isn’t there to accept electrons (for instance, because
a person is not breathing in enough oxygen), the electron
transport chain will stop running, and ATP will no longer be
produced by chemiosmosis.
» Without enough ATP, cells can’t carry out the reactions they
need to function, and, after a long enough period of time, may
even die.
 Oxidative phosphorylation
» The electron transport chain is a series of proteins and organic
molecules found in the inner membrane of the mitochondria.
» Electrons are passed from one member of the transport chain to
another in a series of redox reactions.
» Energy released in these reactions is captured as a proton
gradient, which is then used to make ATP in a process called
chemiosmosis.
» Together, the electron transport chain and chemiosmosis
make up oxidative phosphorylation.
Complex I: NADH dehydrogenase complex
Complex II: Succinate dehydrogenase complex
Complex III: Cytochrome bc1 complex
Complex IV: Cytochrome oxidase
 Oxidative phosphorylation
1.Delivery of electrons by NADH and
FADH2
». Reduced electron carriers (NADH and
FADH2) from other steps of cellular
respiration transfer their electrons to
molecules near the beginning of the
transport chain.
» In the process, they turn back into NAD+
and FAD, which can be reused in other
steps of cellular respiration.
 Oxidative phosphorylation

2.Electron transfer and proton pumping.

»As electrons are passed down the chain, they
move from a higher to a lower energy level,
releasing energy.
»Some of the energy is used to pump H+ ions,
moving them out of the matrix and into the
intermembrane space.
»This pumping establishes an electrochemical
gradient.
 Oxidative phosphorylation
3. Splitting of oxygen to form water
• At the end of the electron transport chain, electrons are transferred to
molecular oxygen, which splits in half and takes up H+ to form
water.
4. Gradient-driven synthesis of ATP. As H+ions flow down their
gradient and back into the matrix, they pass through an enzyme called
ATP synthase, which harnesses the flow of protons to synthesize ATP.
 ELECTRON TRANSPORT CHAIN
» ETC is a collection of membrane-embedded proteins and organic
molecules, most of them organised into four large complexes
labeled I to IV.
» In eukaryotes, many copies of these molecules are found in the
inner mitochondrial membrane.
» In prokaryotes, the electron transport chain components are found
in the plasma membrane.
» As the electrons travel through the chain, they go from a higher to
a lower energy level, moving from less electron-hungry to more
electron-hungry molecules.
» Energy is released in these “downhill” electron transfers, and
several of the protein complexes use the released energy to pump
protons from the mitochondrial matrix to the intermembrane
space, forming a proton gradient
» All of the electrons that enter the transport chain come from
NADH and FADH2 molecules produced during earlier stages of
cellular respiration: glycolysis, pyruvate oxidation, and the citric
acid cycle.
NADH is very good at donating electrons in redox reactions
(that is, its electrons are at a high energy level), so it can
transfer its electrons directly to complex I, turning back into
NAD+. As electrons move through complex I in a series of
redox reactions, energy is released, and the complex uses
this energy to pump protons from the matrix into the
intermembrane space.
FADH2 is not as good at donating electrons as NADH (that
is, its electrons are at a lower energy level), so it cannot
transfer its electrons to complex I. Instead, it feeds them
into the transport chain through complex II, which does
not pump protons across the membrane.
» Because of this "bypass," each FADH2 molecule causes fewer
protons to be pumped (and contributes less to the proton
gradient) than an NADH.
» Beyond the first two complexes, electrons from NADH and FADH2 travel
exactly the same route.
» Both complex I and complex II pass their electrons to a small, mobile
electron carrier called ubiquinone (Q), which is reduced to form QH2 and
travels through the membrane, delivering the electrons to complex III.
» As electrons move through complex III, more H+ ions are pumped across the
membrane, and the electrons are ultimately delivered to another mobile
carrier called cytochrome C (cyt C).
» Cyt C carries the electrons to complex IV, where a final batch of H+ ions is
pumped across the membrane.
» Complex IV passes the electrons to O2, which splits into two oxygen atoms
and accepts protons from the matrix to form water.
» 4 electrons are required to reduce each molecule of O2, and 2 water
molecules are formed in the process.
» Regenerates electron carriers. NADH and FADH2

pass their electrons to the electron transport chain, turning back
into NAD+ and FAD. This is important because the oxidized forms
of these electron carriers are used in glycolysis and the citric acid
cycle and must be available to keep these processes running.
» Makes a proton gradient. The transport chain builds a proton
gradient across the inner mitochondrial membrane, with a higher
concentration of H+ in the intermembrane space and a lower
concentration in the matrix. This gradient represents a stored form
of energy, and it can be used to make ATP.
 Chemiosmosis
» Complexes I, III, and IV of the electron transport chain are proton pumps. As
electrons move energetically downhill, the complexes capture the released energy
and use it to pump H+ ions from the matrix to the intermembrane space.
» This pumping forms an electrochemical gradient across the inner mitochondrial
membrane.
» The gradient is sometimes called the proton-motive force, and as a form of stored
energy, kind of like a battery.
» Like many other ions, protons can't pass directly through the phospholipid bilayer of
the membrane because its core is too hydrophobic. Instead, H+ ions can move
down their concentration gradient only with the help of channel proteins that form
hydrophilic tunnels across the membrane.
» In the inner mitochondrial membrane, H+ ions have just one channel
available: a membrane-spanning protein known as ATP synthase.
» Conceptually, ATP synthase is a lot like a turbine in a hydroelectric power plant.
Instead of being turned by water, it’s turned by the flow of H+ ions moving down
their electrochemical gradient. As ATP synthase turns, it catalyzes the addition of a
phosphate to ADP, capturing energy from the proton gradient as ATP.
This process, in which energy from a proton gradient is used to make ATP, is
called chemiosmosis.
More broadly, chemiosmosis can refer to any process in which energy stored in a
proton gradient is used to do work.
Although chemiosmosis accounts for over 80% of ATP made during glucose
breakdown in cellular respiration, it’s not unique to cellular respiration.
Two net ATP are made in glycolysis, and another two ATP (or energetically equivalent
GTP) are made in the citric acid cycle.
Beyond those four, the remaining ATP all come from oxidative phosphorylation.
Based on a lot of experimental work, it appears that four H+ ions must flow back into the
matrix through ATP synthase to power the synthesis of one ATP molecule.
When electrons from NADH move through the transport chain, about 10 H+ ions are
pumped from the matrix to the intermembrane space, so each NADH yields about 2.5
ATP.
Electrons from FADH2, which enter the chain at a later stage, drive pumping of only 6
H+, leading to production of about 1.5 ATP.
ATP yield from NADH made in glycolysis. This is because glycolysis happens in the
cytosol, and NADH can't cross the inner mitochondrial membrane to deliver its electrons to
complex I. Instead, it must hand its electrons off to a molecular “shuttle system” that
delivers them, through a series of steps, to the electron transport chain.
Some cells have a shuttle system that delivers electrons to the transport chain via FADH2.
In this case, only 3 ATP are produced for the two NADH of glycolysis.
Other cells of have a shuttle system that delivers the electrons via NADH, resulting in the
production of 5 ATP.