CHEM 201: BIOCHEMISTRY| LECTURE
CITRIC ACID CYCLE, ELECTRON TRANSPORT CHAIN, AND
OXIDATIVE PHOSPHORYLATION
FINALS | S.Y 2022 – 2023
MICHAEL DANN SUPERIO, MSc
OUTLINE
I. Four Mechanisms
II. The Central Role of Citric Acid Cycle in
Metabolism
A. Pathway of the Citric Acid Cycle
B. Conversion of Pyruvate to Acetyl-CoA
III. Steps in the Citric Acid Cycle
A. Step 1: Formation of Citrate
B. Step 2: Isomerization of Citrate to
Isocitrate
C. Step 3: Formation of α-Ketoglutarate
and CO₂- First Oxidation
D. Step 4: Formation of Succinyl-CoA and
CO₂- Second Oxidation
E. Step 5: Formation of Succinate
II. THE CENTRAL ROLE OF CITRIC ACID CYCLE IN
F. Step 6: Formation of
Fumarate-FAD-Linked Oxidation
G. Step 7: Formation of L-Malate
H. Step 8: Regeneration of Oxaloacetate-
Final Oxidation Step
I. Summary
J. Notes on Citric Acid Cycle
IV. Energetics and Control of the Citric Acid Cycle
V. Citric Acid Cycle: Catabolism and Anabolism
A. Catabolism
B. Anabolism
VI. The Role of Electron Transport in Metabolism
A. Aerobic Metabolism
B. The Importance of Mitochondrial
Structure in ATP Production
VII. Organization of Electron Transport Complexes
A. Complex I: NADH-CoQ Oxidoreductase
B. Complex II: Succinate-CoQ
Oxidoreductase
C. Complex III: CoQH2-Cytochrome C
Oxidoreductase (Cytochrome reductase
D. Complex IV: Cytochrome c Oxidase
VIII. Electron Transport Chain and Phosphorylation
A. Oxidative Phosphorylation
B. Mechanism of Coupling in Oxidative
Phosphorylation
IX. Shuttle Mechanisms
X. Complete Oxidation of Glucose
CITRIC ACID CYCLE, ELECTRON TRANSPORT
CHAIN, AND
OXIDATIVE PHOSPHORYLATION
I. FOUR MECHANISMS
● Four Mechanisms
○ Carbohydrate Metabolism
○ Lipid Metabolism
○ Amino Acid Metabolism
○ Photosynthesis
○ These four mechanisms are ultimately
interlinked with each other through the
citric acid cycle inside the mitochondria
○ It is important for us to understand citric
acid cycle as well as electron transport
chain (ETC) and oxidative
phosphorylation because it allows us to
understand many diseases. Any
disruptions in this cycle will lead to
depletion of energy in certain diseases
METABOLISM
● Citric Acid Cycle
○ One of the important aspects of aerobic
metabolism
■ The evolution of aerobic
metabolism by which nutrients
are oxidized to carbon dioxide
and water was an important
step in the history of life on
Earth
■ All organisms can obtain far
more energy from nutrients by
aerobic oxidation than
anaerobic oxidation
○ First step is the use of oxygen
● Glycolysis
○ Produces only two molecules of ATP
○ Actually it’s four, but the net production
of glycolysis was only two because the
two ATP is reused during the first part of
glycolysis which was the priming phase
● In this lesson, we shall see how 30 to 32
molecules of ATP can be produced from each
molecule of glucose in complete aerobic
oxidation to carbon dioxide and water
○ Free processes play roles in aerobic
metabolism
● Citric Acid Cycle is Amphibolic
○ Plays a role in both catabolism and
anabolism
○ Catabolism
■ Breakdown of nutrients
○ Anabolism
■ Reductive synthesis of
biomolecules
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 ■ Building of nutrients
○ Citric acid cycle is a part of the pathway
of aerobic oxidation of nutrients,
basically the catabolic part, some of the
molecules that are included in this cycle
are actually the starting points of
biosynthetic pathways. You will see the
byproducts of the citric acid cycle are
actually the products that utilized to
begin other metabolic pathways
○ Metabolic pathways operate
simultaneously
● Citric Acid Cycle has two other common names:
○ Krebs Cycle
■ Named after sir Hans Adolf
Krebs who first investigated the
pathway
○ Tricarboxylic Acid Cycle (TCA)
■ Involved in this cycle are
generally acids with three
carboxylic groups
A. PATHWAY OF THE CITRIC ACID CYCLE
● Difference of glycolysis and citric acid cycle
○ Is the part of the cell in which these
pathways occur
○ Glycolysis
■ Occurs in the cytosol in most
eukaryotes
○ Citric Acid Cycle
■ Has 8 steps
■ Occurs in the mitochondria,
specifically in the matrix of the
organelle
■ Most of the enzymes of the
citric acid cycle are present in
the mitochondrial matrix
■ Note: One reaction takes place
in the inner mitochondrial
membrane (FAD-linked
reaction)
● Green background
○ Cytosol
● Mitochondrion is divided into four:
■ Outer Mitochondrial Membrane-
shields or protects the insides of
the mitochondria from the
outside environment of the
organelle
■ Inner Mitochondrial Membrane-
dark pigments in the photo
■ Intermembrane Space- located
between the outer and inner
mitochondrial membrane which
plays a role in the functioning of
the mitochondria; it participates
in oxidative phosphorylation
■ Mitochondrial Matrix- located
inside the inner mitochondrial
membrane where ribosomes
and a lot of enzymes are
floating around
● This the overall reaction of the citric acid cycle
○ One pyruvate is utilized in this cycle
○ There are two pyruvate molecules that
are yielded from one glucose molecule,
so there will be two citric acid cycle will
occur in one molecule of glucose
○ Oxaloacetate has the highest
concentration in the mitochondrial matrix
■ Oxaloacetate is replenished as
citric acid cycle is finished in
releasing cofactors that are
used in the electron transport
chain
The Reactions of the Citric Acid Cycle
Step Reaction Enzymes
1 Acetyl CoA + Citrate synthase
Oxaloacetate +H₂O →
Citrate + CoA-SH
2 Citrate → Isocitrate Aconitase
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 ● Coenzyme A
3 Isocitrate + NAD⁺ → Isocitrate
α-Ketoglutarate + NADH dehydrogenase ○ Contains an SH group
+ CO₂ + H⁺

4 α-Ketoglutarate + NAD⁺ α-Ketoglutarate

+ CoA-SH → dehydrogenase
Succinyl-CoA + NADH +
CO₂ + H⁺

5 Succinyl-CoA +GDP + Pᵢ Succinyl-CoA

→ Succinate + GTP + synthetase
CoA-SH

6 Succinate + FAD → Succinate

Fumarate + FADH₂ dehydrogenase
7 Fumarate + H₂O → Fumarase
L-Malate
8 L-Malate + NAD⁺ → Malate
Oxaloacetate + NADH + dehydrogenase
H⁺
B. CONVERSION OF PYRUVATE TO ACETYL-CoA
● This is the first part before we enter the main
citric acid cycle
● Pyruvate does not enter the cycle directly, it is
converted first to acetyl coenzyme by series of
reactions
● Pyruvate can come from several sources
including glycolysis; it moves around the cytosol
into the mitochondrion via a specific transporter
which is present along the mitochondrial
membrane
● Transport protein is an enzyme complex
○ Inside the transport protein is an
enzyme system called the pyruvate
dehydrogenase complex
● Pyruvate Dehydrogenase Complex
○ Responsible for the conversion of
pyruvate to carbon dioxide
○ Attaches the acetyl portion of acetyl
coenzyme A
● There’s a reduction process involved; There’s
an involvement of NAD which is actually an
energy coupling pathway
● Photo Interpretation:
○ The S—CoA is part of the Coenzyme A
○ HS —- CoA
■ The SH or basically the H part
is released
● The carbon dioxide at the left side of the photo
which is highlighted in gray color is also released
● C=O
|
CH3
○ This will attach to the S in HS —- CoA,
hereby resulting to acetyl CoA
● Thiol Group
○ Thiol is an analog of alcohol (OH)
○ Present at one end of the coenzyme
molecule which is at the point at which
acetyl group is attached
■ As a result, coenzyme is
frequently shown in equations
as CoA-SH
● Acetyl Coenzyme is a thioester
○ With the sulfur atom replacing the
oxygen at the usual carboxylic center
which refers to as ester
○ Thioesters are high-energy compounds
■ Hydrolysis of ester releases
enough energy to drive other
reactions; An oxidation reaction
precedes the transfer of the
acetyl group to the coenzyme A
■ This is the oxidation reaction
● The whole process involves several enzymes,
all of which are found in the transport protein
● Enzymes that convert pyruvate to acetyl-CoA:
○ Pyruvate Dehydrogenase (PDH)
○ Dihydrolipolyl Transacetylase

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 ○ Dihydrolipoyl Dehydrogenase III. STEPS IN THE CITRIC ACID CYCLE
■ These first three enzymes is
directly involved in the A. STEP 1: FORMATION OF CITRATE
conversion of pyruvate to
acetyl-CoA
○ Pyruvate Dehydrogenase Kinase
○ Pyruvate Dehydrogenase Phosphatase
■ These two enzymes controls
the pyruvate dehydrogenase
(PDH)
■ Without these two enzymes
there would be no PDH that is
functional to convert pyruvate to
acetyl-CoA

● First step in the citric acid cycle is the formation

of citrate; The enzyme that catalyzes this is the
citrate synthase
● Oxaloacetate which has the highest
concentration inside the mitochondrial matrix
reacts with acetyl coenzyme A plus water; This is
a hydrolysis reaction to yield citrate and replenish
coenzyme A; The coenzyme A at the end of the
reaction is now ready to receive another two
carbon unit from pyruvate; It will now come back
to the transport protein
The mechanism of the pyruvate dehydrogenase reaction ● By the action of citrate synthase will yield citrate
● Step 1: Pyruvate loses CO2 and HETPP is
formed B. STEP 2: ISOMERIZATION OF CITRATE TO
○ Decarboxylation of pyruvate occurs with ISOCITRATE
formation of hydroxyethyl-TPP
■ TPP refers to thiamine
pyrophosphate
● It is a metabolite of
vitamin B1
○ The presence of vitamin B complexes is
important in this step
● Step 2: Hydroxyethyl group is transferred to
lipoic acid and oxidized to form acetyl
dihydrolipoamide
○ Transfer of the two-carbon unit to lipoic
acid
● Isomerization
■ Lipoic acid is part of a complex
○ Simple rearrangement of molecules
which is inside the transport
● Citrate is dehydrated by an enzyme aconitase;
protein
There’s a release of water to yield cis-Aconitate
● Step 3: Acetyl group is transferred to CoA
which is an unstable molecule
○ Formation of acetyl-CoA
● After accepting another water molecule,
● Step 4: Dihydrolipoamide is reoxidized
cis-Aconitate will immediately reconstituted to
○ Lipoic acid is reoxidized in this reaction
isocitrate
○ Involves the simultaneous replenishment
of the lipoic acid
● This process happens all throughout this
shuttling mechanism of pyruvate into the matrix
of the mitochondrion; Once inside the matrix of
the mitochondrion, acetyl CoA is released and
will now participate in the citric acid cycle

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 isocitrate dehydrogenase; Then,
oxalosuccinate is decarboxylated and
the carbon dioxide and α-ketoglutarate is
released
● This is the first of the reactions along the citric
acid cycle in which NADH is produced; One
molecule of NADH is produced from NAD at this
stage by the loss of two electrons in the
oxidation process
● As we saw in the pyruvate dehydrogenase
complex earlier, each NADH produced leads to
the production of 2.5 ATP in later stages of
● Aconitase requires iron aerobic metabolism
○ Fe²⁺ or ferrous iron ● All of the NADH throughout the citric acid cycle
● One of the interesting features of the reaction is will meet during the electron transport chain
that citrate is symmetrical or an achiral
compound; This symmetrical compound is D. STEP 4: FORMATION OF SUCCINYL-CoA and CO₂-
converted to isocitrate which is a chiral SECOND OXIDATION
compound; From achiral it becomes chiral that
cannot be superimposed on its mirror image. It is
often possible for a chiral compound of several
different isomers and isocitrate has four possible
isomers, but only one of the four is produced by
this reaction
● Aconitase, the enzyme that catalyzes the
conversion of citrate to isocitrate can select one
end of the citrate molecule in preference to the
other. This is what we call high affinity to a
certain molecule

C. STEP 3: FORMATION OF α-KETOGLUTARATE and ● The second oxidative decarboxylation takes

CO₂– FIRST OXIDATION
place in step four of the citric acid cycle in which
the carbon dioxide and succinyl coenzyme A are
formed from α-ketoglutarate and coenzyme A
● Remember, coenzyme A is released during the
first step; So that coenzyme A will either shuttle
back towards the formation of acetyl coenzyme A
or it could be utilized in this fourth step along the
citric acid cycle; This reaction is similar to the
one which acetyl coenzyme is formed from
pyruvate with NADH produced from NAD; Each
NADH eventually gives rise to 2.5 ATP; 2.5
multiplied to two because there are two pyruvate
molecules from one glucose
● The third step the oxidative decarboxylation of
● The reaction occurs in several stages and is
isocitrate
catalyzed by the enzyme system called the
● Decarboxylation
alpha ketoglutarate dehydrogenase complex
○ The removal of carbon through the form
which is similar to the pyruvate dehydrogenase
of a carbon dioxide
complex; Each of these multi-enzyme consists of
● Decarboxylation of isocitrate to α-ketoglutarate
three enzymes that catalyze the overall reaction;
and carbon dioxide is the first of two oxidative
The reaction takes place in several steps; There
decarboxylation of the citric acid cycle
is a requirement of thiamine pyrophosphate,
● The enzyme that catalyzes this is the isocitrate
FAD or flavin adenine nucleotide, lipoic acid, and
dehydrogenase
magnesium
● The reaction takes place in two steps:
○ Magnesium ions are not exactly involved
○ First, the isocitrate is oxidized; There’s a
in the chemical reaction of most
removal of hydrogen to oxalosuccinate
energy-rich compounds; Mg²⁺ is there to
which remains bound to the enzyme,
stabilize the structure

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 ■ For example ATP is a very
electronegative ion and very
rich in electrons, to stabilize
those electrons, it needs an
inert molecule such as
magnesium to stabilize its
structure; Otherwise, the
phosphate at the terminal end
of the ATP will be released
immediately
● This is reaction is highly exergonic and as is the
one catalyzed by pyruvate dehydrogenase which
means it is spontaneous
● The two molecules of carbon dioxide have been
produced by the oxidative decarboxylations of
the acetic acid; The removal of the carbon
dioxide makes the citric acid irreversible in vivo
although invitro which separates reaction is
reversible
● The NADH will go to electron transport
● This step is the second time the carbon dioxide is
formed
E. STEP 5: FORMATION OF SUCCINATE
● Succinate is now used to form fumarate
● Remember, I mentioned earlier that one of the
reactions in volume citric acid cycle does not
occur in the mitochondrial matrix, but inside the
inner mitochondrial membrane, from there, we
will see the final stages of citric acid cycle
● Succinate is attached to succinate
dehydrogenase
○ The succinate dehydrogenase is the
FAD linked reaction; It is attached to one
of the complexes for electron transport
chain
○ Succinate dehydrogenase is an enzyme;
Instead of floating around the
mitochondrial matrix, it is bound to the
inner mitochondrial membrane, one of
the complexes involved in the electron
transport chain
● Fumarase will be used to form L-malate then
malate dehydrogenase will reform oxaloacetate

F. STEP 6: FORMATION OF FUMARATE-FAD-LINKED

OXIDATION

● Succinyl coenzyme A is converted to succinate

● Thioester
○ Refers to the sulfur
● The succinyl coenzyme A is hydrolyzed to
produce succinate
● There is a release of energy; GDP or guanosine
diphosphate to form guanosine triphosphate, so
there is an addition of energy; GTP is an analog
of ATP; It is basically the same but the
nitrogenous base is just different, instead of
adenine, it is guanine
● The enzyme that catalyzes this reaction is
succinyl coenzyme A or succinyl CoA synthetase ● Succinate is oxidized by FAD
to yield succinate ○ Two electrons are removed from
succinate
● FADH₂ contains electrons and will enter the
electron transport chain together with the NADH
earlier

G. STEP 7: FORMATION OF L-MALATE

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 ● After forming fumarate, it will be released into the
mitochondrial matrix; Then it is hydrated to
L-malate; There is a stereospecificity in the
reaction and malate has two enantiomers L and
D, but only L-malate is produced
● The enzyme that catalyzes this reaction is
fumarase
H. STEP 8: REGENERATION OF OXALOACETATE-
FINAL OXIDATION STEP
● L-malate releases an electron, basically it is
received by NAD⁺ to reform oxaloacetate plus
NADH plus a proton molecule or hydrogen ion
molecule
● The enzyme that catalyzes this reaction is
malate dehydrogenase
● Oxaloacetate being a precursor to the start of the
citric acid cycle is reused or it could be
transformed
● Remember, in gluconeogenesis, oxaloacetate
must be first converted to malate, so it will follow
a series of all of those reactions to be reformed
to become L-malate; L-malate is shuttled outside
the mitochondria to be oxidized once more to
become oxaloacetate and then oxaloacetate to
pyruvate; Pyruvate now will be used for
gluconeogenesis
● All of these reactions are simultaneous as long
as one of the products are formed they will enter
immediately the next reaction
● You must remember the reactions and the points
where NADH and FADH₂ are
● The Q in QH₂ is accompanied by FADH₂. This is
part of the electron transport chain
I. SUMMARY
● This is the summary of the products that are
formed under citric acid cycle
● Note: 2 net ATP were produced per glucose unit
during glycolysis, and two NADH, which will give
rise to another five ATP: a total of 7 ATP
● 25 ATP (Krebs and ETC) + 7 ATP (glycolysis) =
32 ATP/glucose molecule
J. NOTES ON CITRIC ACID CYCLE
● Most important reactions are identified by those
that have important cofactors (NADH, FADH₂,
GTP)
● Also important steps are those where CO₂ is
given off
● One purpose of the cycle is to produce energy
○ Does so by producing GTP directly (an
analog of ATP)
○ Also produces energy indirectly by
producing reduced electron carriers
(NADH, FADH₂)
■ It directly yields energy and
indirectly yields products that
are used to generate ATP
● The decarboxylation mean that for every three
carbons entering a pyruvate, three carbons are
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 effectively lost during the cycle, a fact which has ○ The pyruvate dehydrogenase is inhibited
many implications to our metabolism by ATP acetyl coenzyme A and NADH,
that is if there is a high concentration of
IV. ENERGETICS AND CONTROL OF THE CITRIC ATP acetyl coenzyme A and NADH;
ACID CYCLE These three molecules becomes
● The reaction of pyruvate to acetyl-CoA is allosteric inhibitors of pyruvate
exergonic dehydrogenase via the pyruvate
○ Meaning it releases, it’s spontaneous; dehydrogenase kinase and pyruvate
It’s also exothermic because it releases dehydrogenase phosphatase; By
energy inhibiting those two enzymes, pyruvate
● Citric acid cycle itself is exergonic dehydrogenase is not activated and
○ One reaction however, is endergonic therefore, these shuttling mechanism is
■ Oxidation of malate to shut
oxaloacetate ● How is control exerted within the citric acid
■ The release of GPP, that cycle?
particular reaction is neutralized
by the overall energy release of
citric acid
■ The overall reaction is part of a
pathway that releases energy
and it is not surprising that the
enzyme that initiates it is
inhibited by ATP and NADH
because both compounds are
abundant when a cell has a
good deal of energy readily
○ Remember, the enzyme that catalyzes
available; So, if there is a high
the conversion of oxaloacetate
concentration of ATP and
coenzyme A and water to citrate
NADH, citric acid cycle can be
dehydrogenase, that enzyme is inhibited
inhibited
by ATP, NADH, succinyl-CoA, and
■ The end products of a series of
citrate
reactions inhibit the first
■ If there is a high concentration
reaction of the series, and the
of these four molecules, citrate
intermediate reactions should
dehydrogenase is inhibited
not face when their products are
○ Isocitrate dehydrogenase is inhibited
not needed
by high levels of ATP and NADH
● Control of the citric acid cycle is exercised at
■ ATP and ADP are basically
three points
almost the same, they belong to
○ Three enzymes within the citric acid
one equation; If ATP levels are
cycle play a regulatory role
high, you are inhibiting
○ There is also control of access to the
isocitrate dehydrogenase;
cycle via pyruvate dehydrogenase
However, if ADP is high
compared to ATP, then it
stimulates the isocitrate
dehydrogenase as well as
NADH
○ α-ketoglutarate is inhibited by ATP,
NADH, and succinyl-CoA
■ If the cell has high levels of
these compounds then basically
citric acid cycle will be inhibited
because what’s the purpose of
releasing energy-rich molecules
● Control points in the conversion of pyruvate to
if there is a high concentration
acetyl-coA and in the citric acid cycle
of molecules that contains high
● How does pyruvate dehydrogenase reaction
energy, it’s counterproductive
control the citric acid cycle?

V. CITRIC ACID CYCLE: CATABOLISM AND

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 ANABOLISM cross the mitochondrial membrane and
participate in the citric acid cycle
A. CATABOLISM ● Sugars, fatty acids, and amino acids are all
included in these overall catabolic scheme;
Basically, citric acid cycle is a general catabolic
pathway
● Citric acid cycle is amphibolic, so it participates
in both catabolism and anabolism

B. ANABOLISM

● These shows the various catabolic pathways that

feed into the citric acid cycle
● A summary of catabolism, showing the central
role of the citric acid cycle. Note that the end
products of the catabolism of carbohydrates,
lipids, and amino acids all appear. (PEP is
phosphoenolpyruvate; a-KG is α-ketoglutarate;
TA is transamination; is a multistep pathway)
● The nutrients taken in by an organism can
include large molecules and this observation is
especially true in the case of animals which
ingests polysaccharides, proteins, and lipids
● How mammals keep an adequate supply of
which are polymers
metabolic intermediates: An anabolic reaction
○ For example, humans; Nucleic acid
uses a citric acid cycle intermediate
constitutes a very small percentage of
(a-ketoglutarate is transaminated to glutamate in
the nutrients present in foods and we
our example), competing with the rest of the
shall not consider therefore catabolism
cycle. The concentration of acetyl-CoA rises and
● The first step in the breakdown of nutrients is
signals the allosteric activation of pyruvate
the degradation of large molecules to smaller
carboxylase to produce more oxaloacetate
ones (Mastication → mechanical digestion →
○ Anaplerotic reaction
chemical digestion) via enzymatic pathways
■ Replenishes a citric acid cycle
inside your digestive system; Once
intermediate
polysaccharides are hydrolyzed by specific
○ Part of glyoxylate pathway
enzymes to produce sugar monomers; An
● The citric acid cycle is a source of starting
example is a breakdown of starch by amylases
materials for the biosynthesis of many important
and lipases hydrolyze triacylglycerols to give
biomolecules
fatty acids and glycerol; Proteins are digested by
● The supply of the starting materials that are
proteases with amino acids as the end product;
components of the cycle must be replenished if
Sugar, fatty acids, amino acids then enter the
the cycle is to continue operating
specific catabolic pathways
● Oxaloacetate
● Glycolytic pathway is the pathway by which
○ In an organism, must be maintained at a
sugars are converted to pyruvate which then
level sufficient to allow acetyl coenzyme
enters the citric acid cycle
to enter the cycle
● These different amino acids can also enter the
○ In some organisms some acetyl-CoA
citric acid cycle by any of these points
can be directly converted to
○ Amino acids can enter various pathways
oxaloacetate or other citric acid cycle
○ Some of them converted to acetyl-CoA,
intermediates
some are converted to one of the
■ Mammals cannot do this;
products along the cycle
Humans cannot do this
● Citric acid cycle takes place in the mitochondria
and many of the end products of catabolism

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 ○ In mammals, it is produced from
pyruvate by the enzyme pyruvate
carboxylase
■ Acetyl-CoA can be used
through our allosteric activation
of pyruvate carboxylase.
Oxaloacetate by pyruvate
● Reaction which produces oxaloacetate for
pyruvate provides a connection between the
amphibolic citric acid cycle and the anabolism
sugars via gluconeogenesis
● Pyruvate cannot be produced by acetyl-CoA in
mammals because acetyl-CoA is the end product
of catabolism of fatty acids
○ Mammals could not exist with fats or
acetate as the sole carbon source
■ You cannot eliminate
carbohydrate in your diet
because of this pathway
■ Without carbohydrates,
pyruvate cannot be yielded
● Carbohydrates are the principal energy and
carbon source in animals
○ Glucose is especially critical in humans
because it is the preferred fuel of our
brain cells
○ In plants however, they can carry out the
conversion of acetyl-CoA to pyruvate
and oxaloacetate
■ Plants can exist without
carbohydrates
● This is what makes Krebs cycle anabolic in
nature
● Transfer of the starting materials of
gluconeogenesis from the mitochondrion to the
cytosol. Note that phosphoenolpyruvate (PEP)
can be transferred from the mitochondrion to the
cytosol, as can malate. Oxaloacetate is not
transported across the mitochondrial membrane.
(1 is PEP carboxykinase in mitochondria; 2 is
PEP carboxykinase in cytosol)
○ Oxaloacetate should follow the cycle to
become malate and then malate can exit
the mitochondrion which malate is
oxidized to reform oxaloacetate then
converted to pyruvate via
phosphoenolpyruvate in a pathway of
gluconeogenesis
VI. THE ROLE OF ELECTRON TRANSPORT IN
METABOLISM
A. AEROBIC METABOLISM
● Aerobic Metabolism
○ Utilizes oxygen; highly efficient way for
an organism to extract energy from
nutrients
○ Includes conversion of pyruvate to acetyl
CoA, citric acid cycle, and electron
transport occur in the mitochondria;
glycolysis (anaerobic process) takes
place in the cytosol
○ Electron transport: involves a series of
transfer of electrons from coenzymes
(NADH, FADH2) to the final acceptor,
oxygen
○ In eukaryotic cells, the aerobic
processes all occur in the mitochondria
○ Aerobic processes
■ Conversion of pyruvate to
acetyl-CoA
■ Citric acid cycle
■ Electron transport
○ Glycolysis does not need an input of
oxygen
■ Anaerobic process
■ Takes place is the cytosol
● Oxygen in metabolism is the final acceptor of
electrons in the electron transport chain
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● Electron transport occurs inside the inner
mitochondrial membrane
○ Involves the production of ATP, water,
and CO2
○ ETC is coupled with oxidative
phosphorylation to produce ATP, water,
and CO2
● Reactions of electron transport chain takes place
in the inner mitochondrial membrane
○ Electron Transport Chain; Mitochondrial
membrane
○ Citric Acid Cycle; Mitochondrial matrix
B. THE IMPORTANCE OF MITOCHONDRIAL
STRUCTURE IN ATP PRODUCTION
● A proton gradient is established across the inner
mitochondrial membrane as a result of electron
transport. Transfer of electrons through the
electron transport chain leads to the pumping of
protons from the matrix to the intermembrane
space. The proton gradient (also called the pH
gradient), together with the membrane potential
(a voltage across the membrane), provides the
basis of the coupling mechanism that drives ATP
synthesis
● In general, the energy released by the oxidation
of nutrients is used by organisms in the form of
chemical energy of Adenosine Triphosphate
(ATP)
● The production of ATP in the mitochondria is the
result of oxidative phosphorylation
○ Not because of Electron Transport Chain
■ Coupled
● Oxidative Phosphorylation involves the
phosphorylation of ADP to ATP
○ The production of ATP is separate from
electron transport to oxygen
○ But the reactions of the electron
transport chain are tightly coupled to the
synthesis of ATP by phosphorylation of
ADP
● The operation of Electron Transport Chain leads
to pumping of protons across the inner
mitochondrial membrane, creating a pH gradient
(Proton gradient)
● Electron Transport Chain (ETC)
○ How NADH and FADH2 establishes the
protein gradient
● Proton Gradient
○ Represent stored potential energy and
provides basis of the coupling
mechanism
○ Mechanism: Chemiosmotic coupling
○ Difference in concentration
○ High Concentration of Hydrogen
■ Low pH
○ Low Concentration of Hydrogen
■ High pH
● Schematic representation of the electron
transport chain, showing sites of proton pumping
coupled to oxidative phosphorylation. FMN is the
flavin coenzyme flavin mononucleotide, which
differs from FAD in not having an adenine
FERNANDEZ, J. & DEAPERA | 2F | 11 of 16
 nucleotide. CoQ is coenzyme Q. Cyt b, cyt c1,
cyt c, and cyt aa3 are the heme-containing
proteins cytochrome b, cytochrome c1,
cytochrome c, and cytochrome aa3, respectively
● Cytochrome B and Cytochrome 1 are the sites of
proton pumping coupled to ATP production
(Oxidative Phosphorylation)
VII. ORGANIZATION OF ELECTRON TRANSPORT
COMPLEXES
● Takes place in the lining of the inner
mitochondrial membrane
● The electron transport chain, showing the
respiratory complexes. In the reduced
cytochromes, the iron is in the Fe(II) oxidation
state; in the oxidized cytochromes, the oxygen is
OXIDOREDUCTASE (CYTOCHROME REDUCTASE)
in the Fe(III) oxidation state
● Complex 1 and Complex 2 are linked to Complex
3 through CoQ,
○ So Complex 1 and Complex 2 are not
simultaneous
■ Independent from each other
■ Both donate electron to CoQ,
electron transported to complex
3 , Complex 3 transfers electron
to Cytochrome C and then
reduce Cytochrome C to
Complex 4
● The oxidized and reduced forms of coenzyme Q.
Coenzyme Q is also called ubiquinone
● CoQ (Oxidized quinone form) is reduced to
CoQH2 (Reduced hydroquinone form) by adding
hydrogen ions
A. COMPLEX I: NADH-COQ OXIDOREDUCTASE
● Transfers the electrons from NADH to coenzyme
Q (CoQ)
○ NADH + H+ + E-FMN → NAD+ +
E-FMNH2 E-FMNH2 + 2Fe-Soxidized →
E-FMN + 2Fe-Sreduced + 2H+ 2Fe-Sreduced
+ CoQ + 2H+ → 2Fe-Soxidized + CoQH2
○ NADH + H+ + CoQ → NAD+ + CoQH2
● Catalyzes the first step of electron transport
namely the transfer of electrons from NADH
along the mitochondrial membrane
● This complex is an integral part of the inner
mitochondrial membrane
○ Integral; Bypasses the lipid bilayer
○ Includes among other subunits several
proteins that contain an iron sulfur
cluster and flavoprotein that oxidizes
NADH
B. COMPLEX II: SUCCINATE-COQ
OXIDOREDUCTASE
● Transfers the electrons from succinate (in the
form of FADH2) to CoQ
○ Succinate + E—FAD → Fumarate +
E—FADH2 E—FADH2 + Fe—Soxidized →
E—FAD + Fe—Sreduced Fe—Sreduced +
CoQ + 2H+ → Fe—Soxidized + CoQH2
○ Succinate + CoQ → Fumarate + CoQH2
● After this process, CoQH2 (Hydroquinone) will
then pass electrons to complex 3
C. COMPLEX III: COQH2-CYTOCHROME C
● Catalyzes the oxidation (receives the electron) of
reduced coenzyme Q (CoQH2). Then passes the
electron to cytochrome c in a multistep process.
○ CoQH2 + 2 Cyt c[Fe(III)] → CoQ + 2 Cyt
c[Fe(II)] + 2H+
● Flow of electrons from reduced CoQ is not a
simple, direct path. CoQ exists in three forms:
oxidized (CoQ), reduced (CoQH2), and
intermediate (semiquinone, CoQH-). This cycling
of CoQ forms is known as Q cycle.
● CoQ is a lipid draft, a protein complex that is
floating around in the inner mitochondrial
membrane
○ Not just one but many
● Complex 1 and Complex 2 has an efficient way
of transferring electrons
● If one CoQ system if occupied then another CoQ
system will be entertained until it reaches
Complex 3 which will now transfer electron to
Complex 4
● After this process, H+ is transferred to Complex 4
D. COMPLEX IV: CYTOCHROME C OXIDASE
● Catalyzes the transfer of electrons from
cytochrome C to oxygen.
○ 2 Cyt c[Fe(II)] + 2H+ + ½ O2 → 2 Cyt
c[Fe(III)] + H2O
● Complex 3 is Cytochrome reductase while
Complex 4 is Cytochrome oxidase
● Basically your electron from NADH and FADH2 in
the form of hydrogen transfers from one Complex
then CoQ then another Complex 3 then to
Cytochrome C then to Cytochrome C Oxidase
FERNANDEZ, J. & DEAPERA | 2F | 12 of 16
● How it looks like
● The compositions and locations of respiratory
complexes in the inner mitochondrial membrane,
showing the flow of electrons from NADH to O2.
Complex II is not involved and not shown. NADH
has accepted electrons from substrates such as
pyruvate, isocitrate, α-ketoglutarate, and malate.
Note that the binding site for NADH is on the
matrix side of the membrane. Coenzyme Q is
soluble in the lipid bilayer. Complex III contains
two b-type cytochromes, which are involved in
the Q cycle. Cytochrome c is loosely bound to
the membrane, facing the intermembrane space.
In Complex IV, the binding site for oxygen lies on
the side toward the matrix.
○ For electron transport chain to occur
NADH from the cytosol must enter the
matrix before the transfer of electron
occurs
● The sites of pumping electrons are Complex 1, 3,
and 4
VIII. ELECTRON TRANSPORT CHAIN AND
PHOSPHORYLATION
A. OXIDATIVE PHOSPHORYLATION
● Oxidative Phosphorylation
○ Addition of phosphate group to existing
ADP molecules in the inner
mitochondrial membrane.
■ Forms ATP through an enzyme
powered by the chemical
gradient of the mitochondria.
○ Coupling factor that links oxidation and
phosphorylation, and enzyme known as
ATP synthase.
■ Consist of three different kinds
of polypeptide chains (a, b, and
c)
● A model of the F1 and F0 components of the
ATP synthase, a rotating molecular motor. The a,
b, α, β, and δ subunits constitute the stator of the
motor, and the c, γ, and ε subunits form the rotor.
Flow of protons through the structure turns the
rotor and drives the cycle of conformational
changes in α and β that synthesize ATP
B. MECHANISM OF COUPLING IN OXIDATIVE
PHOSPHORYLATION
● Two mechanisms of coupling were proposed
○ Chemiosmotic coupling: involves the
establishment of proton gradient. The
difference in the concentration of
hydrogen drives the ATP synthase to
turn, thus fueling the rotation of the
enzyme.
■ Like a Dynamo or Hydroelectric
Dam. High hydrogen
concentration will flow along the
gradient through ATP synthase
○ Conformational coupling: the proton
gradient is indirectly related to ATP
production; proton gradient leads to
conformational changes in the ATP
thereby releasing the tightly bound ATP
from the synthase
■ ATP synthase can actually
couple ADP and inorganic
phosphate but ATP cannot
escape the grip of ATP
synthase
● The creation of a proton gradient in
chemiosmotic coupling. The overall effect of the
electron transport reaction series is to move
protons (H+) out of the matrix into the
intermembrane space, creating a difference in pH
across the membrane
FERNANDEZ, J. & DEAPERA | 2F | 13 of 16
● Complex 1 can pump out 4 hydrogen molecules.
Complex 3 can pump out 4 hydrogen molecules.
While Complex 4 can pump out 2 hydrogen
molecules

● This is the Chemiosmotic Coupling

● Formation of ATP in the chemiosmotic coupling
accompanies the flow of protons back into the
mitochondrial matrix.
● In this coupling, the amount of hydrogen in the
intermembrane space is increased by the
pumping of the motors of the complexes
● Hydrogen will flow through the ATP synthase
which causes the rotation of the ATP synthase
and drives the attachment of ADP to phosphate ● This is the Conformational Coupling
thereby yielding ATP ● The role of conformational change in releasing
the ATP from ATP synthase. According to the
binding change mechanism, the effect of the
proton flux is to cause a conformational change
that leads to the release of already formed ATP
from ATP synthase
● ATP synthase is able to connect inorganic
phosphate to adenosine diphosphate
○ However ATP once formed cannot
esca[e the grip of ATP synthase

IX. SHUTTLE MECHANISMS

● Shuttling of Important Metabolites through the
Mitochondria
○ NADH from glycolysis cannot cross the
inner mitochondrial membrane to enter
ETC
■ Therefore carriers that can
cross the membrane are used
■ These carriers get NADH and
accompanies it like a shuttle
bus then it’s free to go
○ The number of ATP molecules
generated depend on the nature of
these carriers
■ Varies in the type of cell in
which it occurs

FERNANDEZ, J. & DEAPERA | 2F | 14 of 16

 ● This shuttle uses the fact that malate can cross
the mitochondrial membrane while oxaloacetate
cannot
● The transfer of electrons from NADH in the
cytosol produces NADH in the mitochondrion.
The cytosol oxaloacetate is reduced to malate by
the cytosolic malate dehydrogenase
accompanied by the oxidation of cytosolic NADH
to NAD and the malate then after it crosses the
mitochondrial membrane, it is converted back to
oxaloacetate and is catalyzed by mitochondrial
dehydrogenase and then it follows a series of
reaction to become l-Aspartate which can also
cross the mitochondrial membrane and aspartate
is converted to oxaloacetate in the cytosol
completing the cycle of reactions. Then NADH is
produced in the mitochondrion that passes
electrons so the electron transport chain.
● With the malate-aspartate shuttle, 2.5 moles of
ATP are produced for each mole of cytosolic
● The glycerol–phosphate shuttle NADH
● Uses the presence of the outer face of the inner
mitochondrial membrane but by an FAD X. COMPLETE OXIDATION OF GLUCOSE
dependent enzyme that oxidizes glycerol
phosphate
● Then the glycerol phosphate is produced by the ATP Yield per
Glucose
reduction of the dihydroxyacetone phosphate in
the course of the reaction NADH is oxidized to Pathway Glycerol Malate- NADH FADH2
NAD -Phosph Aspart
○ In this reaction the oxidizing agent which ate ate
is itself reduced is FAD and the product Shuttle Shuttle
is FADH2
○ The FADH2 then passes electrons Glycolysis glucose to pyruvate (Cytosol)
through the electron transport chain
Phosphorylation -1 -1
leading to the production of 1.5 moles of of glucose
ATP for each mole of cytosolic NADH
● This mechanism has also been observed in Phosphorylation -1 -1
mammalian muscle and brain of
● Utilizes the outer face fructose-6-phosp
● Yield 1.5 ATP hate

Dephosphorylatio +2 +2
n of 2 molecules
of 1,3-BPG

Dephosphorylatio +2 +2
n of 2 molecules
of PEP

Oxidation of 2 +2
molecules of
glyceraldehyde-3-
phosphate yields
2 NADH

Pyruvate conversion to acetyl-CoA (Mitochondria)

● The malate–aspartate shuttle
2 NADH +2
● More complex and more efficient mechanism produces
which has been found in mammalian kidney,
liver, and heart Citric acid cycle (Mitochondria)

FERNANDEZ, J. & DEAPERA | 2F | 15 of 16

2 molecules of +2 +2
GTP from 2
molecules of
succinyl-CoA

Oxidation of 2 +6
molecules each
isocitrate,
⍺-ketoglutarate,
and malate yields
6 NADH

Oxidation of 2 +2
molecules of
succinate yields 2
FADH2

Oxidative phosphorylation (Mitochondria)

2 NADH from +3 +5 -2
glycolysis yields
1.5 ATP each if
NADH is oxidized
by
glycerol-phosphat
e shuttle; 2.5 ATp
by
malate-aspartate
shuttle

Oxidative +5 +5 -2
decarboxylation
of 2 pyruvate to 2
acetyl-CoA: 2
NADH produce
2.5 ATP each

2 FADH2 from +3 +3 -2
each citric acid
cycle produce 1.5
ATP each

6 NADH from +15 +15 -6

citric acid cycle
produce 2.5 ATP
each

Net Yield +32 +32 0 0

FERNANDEZ, J. & DEAPERA | 2F | 16 of 16