Citric acid cycle &

electron transport
chain
Recap
• Pyruvate enters into different metabolic reactions

• Pyruvate can be converted into:

✓ Ethanol

✓ Lactic acid

✓ Acetyl CoA

✓ oxaloacetate

• Acetyl CoA and oxaloacetate are produced via aerobic respiration

• Ethanol and lactate are produced during anaerobic respiration

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1.0 Conversion to ethanol

• Occurs predominantly in yeasts

• Is a 2 step reaction:
i. Pyruvate decarboxylase decarboxylates pyruvate into acetylaldehyde

ii. Acetylaldehyde is reduced to ethanol by alcohol dehydrogenase

• Employed in industry to produce breads and ethanol

 2.0 Conversion to lactate

• Carried out by Lactobacillus which are employed to produce yoghurt, buttermilk,

cheese

• Also carried out by our muscles when oxygen supply is less than demand

• Converts 3C pyruvate to 3C lactate

• NAD+ is generated allowing continued generation of ATP

• Muscle fatigue and pain are associated with accumulation of lactate ions
In the presence of oxygen…..
• Aerobic respiration proceeds

• Pyruvate is transported into the mitochondria

• In the mitochondria, pyruvate is transformed into a 2C compound

• A molecule of CO2 is removed

• The resultant compound is called acetyl CoA

 Citric acid cycle
A K A K R E B ’ S C YC L E / T R I C A R B O X Y L I C
A C I D C YC L E
• 2nd step of aerobic respiration

• Transfers energy from pyruvic

acid to electron carriers

• Electron carriers in turn

transfer energy to the ETC
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 Step 1: Formation of citric acid
• Catalysed by citrate synthase

• Condensation reaction joining acetyl CoA (2C) to oxaloacetate (4C)

• Yield is citric acid (6C) and CoA

• Oxaloacetate first condenses to form citryl CoA

• Citryl CoA is hydrolysed to citrate and CoA

• Hydrolysis of the high thioester intermediate drives the reaction in favour of citrate formation
Step 2: Isomerisation of citrate

• Catalysed by aconitase

• Citrate is converted to isocitrate

• This enables oxidative decarboxylation of the 6 C molecule

• Isomerisation is accomplished first by dehydration

• Hydration immediately follows

• Results in an interchange if H atom for a OH group

Step 3: oxidation and decarboxylation of isocitrate
• Catalysed by isocitrate dehydrogenase

• Isocitrate dehydrogenase is allosterically controlled

• Rate limiting step

• This is the 1st of 4 oxidation reactions

• Oxidation of isocitrate yields an unstable β-ketoacid called oxalosuccinate and NADH

• Subsequent decarboxylation yields CO2 and α-ketoglutarate (5C)

Step 4: Oxidation of α-ketoglutarate

• Catalysed by the enzyme complex α-

ketoglutarate dehydrogenase

• Consists of an E2 and E1
(transuccinylase) complex

• Oxidation yields a 4C molecule which

picks up CoA to form succinyl CoA

• Conversion yields one molecule of

NADH and a molecule of CO2
Step 5: Conversion of succinyl CoA
• Catalysed by Succinyl CoA synthetase
(succinate thiokinase)

• Succinyl CoA has an energy rich thioester

bond

• Hydrolysis of Succinyl CoA is coupled to

phosphorylation of the purine nucleoside
GDP

• Thus energy is transferred from the

thioester bond to GDP

• Yield of reaction is 4C succinate, CoA and

GTP
Step 6: succinate is converted to fumerate
• Catalysed by succinate dehydrogenase

• Fumerate is a 4C sugar

• FADH2 is produced

• Hydrogen acceptor is FAD as free energy is insufficient to reduce NAD+

Step 7: Fumerate conversion to malate
• Catalysed by fumerase

• Hydration reaction

• Stereospecific trans addition of a H atom and an OH group

Step 8: Malate is converted to oxaloacetate

• Catalysed by malate
dehydrogenase

• NAD+ is the hydrogen acceptor

• Yields NADH

• Yields oxaloacetate which re enters

the cycle via condensation with acetyl
Co A
Stoichiometry of Krebs cycle
1. Carbon atoms
2 C atoms enter the cycle (acetyl CoA)
2 C atoms leave the cycle (CO2)

2. Hydrogen atoms
4 Hydrogen atoms leave (3 NADH + 1 FADH2)

3. Energy
One GTP is formed

4. Water
2 molecules of water are consumed
• Synthesis of citrate
• Hydration of fumerate
 Oxidative
phosphorylation
Recap
• 3rd step of aerobic respiration

• Transfers energy from

electron carriers to generate
ATP
 Defining oxidative phosphorylation
• NADH and FADH2 are released from cell metabolic pathways

• these pathways include glycolysis, fatty acid oxidation and the citric acid cycle

• Transfer of electrons has a high energy potential

• Thus the pair of electrons trapped in both NADH and FADH2 can be released upon
reduction of another molecule

• Reduction of molecular oxygen therefore frees this energy

• The liberated energy is then trapped in ATP

Oxidative phosphorylation therefore is the process in which ATP is

formed as a result of the transfer of electrons from NADH or FADH2 to
molecular oxygen by electron carriers
Bioenergetics of aerobic respiration
• OP is the culmination of cellular respiration

• It occurs in the mitochondrion

• It describes how energy acquired in metabolic processes is stored as ATP

• Cellular respiration provides energy by:

1. Oxidising carbon fuels in the citric acid cycle to yield electrons with high transfer
potential

2. Electron-motive force is converted into proton-motive force

3. Proton-motive force is converted into phosphoryl transfer potential

• This process is carried out by 3 electron driven proton pumps:

1. NADH-Q oxidoreductase
2. Q-cytochrome c oxidoreductase
3. Cytochrome c oxidase
 Structure of mitochondrion
• Mitochondria have two membrane systems:
i. An outer membrane

ii. An extensive highly folded inner membrane

• The inner membrane is folded into a series of internal ridges called cristae

• The mitochondrion therefore has two parts:

1. The intermembrane space between the outer and inner membranes

2. The matrix which is bound by the inner membrane

• The reactions of the citric acid cycle and fatty acid oxidation occur in the
matrix

• Whereas the reactions of OP occur in the inner mitochondrial membrane

• Cristae increase surface area for OP

• OP is a series of movement of
electrons

• Electrons move down a series of

proteins

• This movement results in expulsion of

H+ across the mitochondrial
membrane

• The last step sees these H+ move back

and therefore generate power to
generate ATP
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Complex 1: NADH Q oxidoreductase
• electrons from NADH enter OP at
NADH-Q-oxidoreductase

• NADH-Q-oxidoreductase is also called

NADH dehydrogenase

• It is a large enzyme 880KD

• It is L shaped

• It has two prosthetic groups:

1. Flavin mononucleotide (FMN)
2. Fe-S centers

• It catalyses the reaction

 • Steps involved in electron transfer in complex

Complex 1 1 are:
i. NADH binds to the vertical arm of NADH-Q-
reductase

ii. Reduces FMN

iii. Electrons flow from FMNH2 to 3 Fe-S complexes

iv. Electrons are transferred to Q

v. Reduction of Q results in uptake of 2 protons in

the matrix

vi. Electrons are transferred to a mobile Q

vii. Release of electrons results in release of

protons on the cystolic side

•Q is also known as ubiquinone

•Q is a mobile reduced form of Coenzyme Q that

transfers electrons to the hydrophobic core of the
membrane
 • Complex II is the point of entry of electrons from

Complex II: Entry of FADH2 FADH2

• Succinate dehydrogenase is responsible for the

generation of FADH2 from succinate in the citric
acid cycle

• Succinate dehydrogenase is part of Complex II

also known as succinate-Q-reductase complex

• FADH2 therefore does not leave the complex

• Instead electrons are transferred to the Fe-S

centers and then to Q for entry into the electron-
transport chain

• Succinate dehydrogenase does not transport

protons

• As a result less energy is formed from the

oxidation of FADH2 than NADH
 • Is the 2nd of the 3 proton pumps of the ETC

Complex III • Also called Q-cytochrome c oxidoreductase or cytochrome

reductase

• A cytochrome is an electron-transferring protein that

contains a haem prosthetic group

• Catalyses the transfer of electrons from QH2 to oxidised

cytochrome c (cyt c) with the concomitant pumping of
protons out of the mitochondrial matrix

• Cty c is a water soluble protein

• Q-cytochrome c oxidoreductase is a dimer

• Each monomer contains 11 subunits

• It contains 3 heme groups

• It also contains 2Fe-S centres

• It contains 2 distinct binding sites for ubiquinone, Q0 and

Q1
 Complex III
• The Q cycle

• Refers to the coupling of electron transfer from Q to cty c

through to transmembrane proton transport

• Cycle facilitates switch from a 2 electron carrier (Q) to a one

electron carrier ( cyt c)

1. QH2 binds to the Q0 site 6. The electron reduces ubiquinone bound to Qi site (Q-)
•Electrons are transferred one at a time
7. A 2nd QH2 binds to the Qo site and is reduced in the same
•Oxidation of QH releases protons into the cytosolic side of
manner
the membrane
8. Q- in the Qi site is now reduced by another electron
2. Q is released and is free to join the ubiquinone pool

3. One electron flows first to 2Fe-2S then to cyt c1 9. Reduced quinone at Qi take up protons from the matrix
contributing to creation of a proton gradient
4. Cty c is converted to its reduced form and diffuses from
the proton pump • At the end of the Q cycle: 2 molecules of QH2 are oxidised, 2

5. 2nd electron is transferred to bL, then bH and finally the Qi molecules of cytochrome C are reduced, 4 protons are released
site on the intermebrane side, 2 protons are removed from the
mitochondrial matrix
Complex IV
• Also known as cytochrome c oxidase

• Consists of 13 subunits (polypeptide chains)

•3 Cu ions arranged as two Cu centers designated A
and B
•CuA/CuA consists of two copper ions linked by two
binding cysteine residues
•Center initially receives electrons from cyt c
•CuB is co-ordinated by 3-histidine residues, one
which is covalently linked to a tyrosine residue
•The two heme A molecules (heme a and heme a3) • Heme a carries electrons from CuA/CuA
have distinct properties and are located in different • Heme a3 passes electrons to CuB
environments in the enzyme • Heme a3 and CuB form the active site at
which O2 is reduced to H2O
Complex IV steps vii. The peroxide forms a bridge between Fe3+
in heme a3 and CuB2+

i. Reduced cyt c transfers an electron to viii. Another cyt c is oxidised and a proton
CuA/CuA taken up, this results in cleavage of the O-
O bond to yield a ferryl group (Fe4+=O) to
ii. Electron moves to heme a3 and finally to
heme a3 and CuB2+-OH
CuB
ix. A final cyt c is oxidised and a 2nd proton
iii. CuB is reduced to cuprous form Cu3+
reduces the ferryl group to Fe3+-OH
iv. Another cyt c is oxidised and a 2nd electon
x. Two additional protons are taken up
moves to heme a3
resulting in the release of two molecules of
v. Heme a3 is now reduced to Fe2+ which is water
able to bind O2
xi. The enzyme returns to its initial oxidised
vi. Reduced CuB, reduces O2 to a peroxide form
(O22-)
Complex IV steps
Summary of OP 1. Complex I : NADH-Q oxidoreductase
1. A flavoprotein
2. Proton pump 1
3. Oxidation of NADH

2. Complex II: Succinate-Q-reductase

• oxidation of FADH2

3. Complex III: Q-cytochrome reductase

• Reduction of cytochrome c
• Proton pump

4. Complex IV: Cytochrome-c-oxidase

• Reduction of molecular oxygen
• Proton pump
Coupling of electron transport and ATP synthesis
• Flow of electrons rom NADH to O2 is an exogernic process

• Synthesis of ATP is an endogernic process

• Coupling occurs via the generation of a proton gradient across the inner membrane of the
mitochondrion

• This gradient creates a proton-motive force that drives ATP synthesis

• The idea was first proposed by Peter Mitchell in 1961 and is termed chemiosmotic hypothesis
ATP Synthase catalyses synthesis of
ATP
• ATP Synthase catalyses synthesis of ATP

• It is also called mitochondrial ATPase or F1F0 ATPase or Complex IV

ATP Synthase structure
• A large membrane-embedded enzyme

• Looks like a ball on a stick

• Consists of an F0 and an F1 subunit

• F1 protrudes into the mitochondrial matrix

• F1 is the ball of ATPase synthase

• F1 contains the catalytic activity of the

synthase
ATP Synthase structure • F1 consists of 5 types of polypeptide
chains (α3β3γδε)

• The α and β subunits comprise the bulk of the

F1 subunit

• They are arranged in a hexameric ring

• Both the α and β chains bind nucleotides but

only the β subunit has catalytic activity

• The central stalk consist of the γ and ε

subunits

• The γ subunit breaks the symmetry of the

hexameric ring facilitating ATP synthesis
 • F0 subunit is the hydrophobic
ATP Synthase structure transmembrane segment

• F0 contains the proton channel

• The proton channel is made of a ring

consisting of 10-14 subunits embedded in
the membrane

• It has an a subunit outside the ring

• F0 and F1 are connected in two ways:

1. By the central γε stalk

2. An exterior subunit consisting of one a

subunit, two b subunits and the δ subunit
 • 3 active sites of Fi take turns to catalyse ATP synthesis

ATP Synthase • The β subunit can take 3 different conformations:

function i. β-ADP

ii. Β-ATP

iii. Β-empty

• A given subunit starts in the β-ADP conformation which binds ADP

and Pi

• Subunit conformation brings about a ready equilibrium between

ADP + Pi and ATP

• Subunit has low affinity for ATP, ATP is readily released and the

• Lies perpendicular to the membrane subunit returns to β-empty

• ATP synthase consists of two functional units: • Another ADP binds

1. A moving/rotor unit consisting of the c ring and
the γε stalk • Conformational change is driven by the passage of protons

2. The stationary unit ( the rest of the enzyme) through the Fo portion of the enzyme

• One complete rotation of the stalk yields 3 molecules of ATP

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Pentose Phosphate pathway