TCA Cycle, Electron Transport Chain,

and Oxidative Phosphorylation

September 23, 2010

Juba University College of Medicine

Lecturer: Chitra Akileswaran

 Announcements

• Biochemistry lectures
– Today: Citric acid cycle, electron transport chain,
and oxidative phosphorylation
– Next Wednesday at 2:00pm: HMP shunt and
glycogen metabolism
– Quiz next Friday covering carbohydrate metabolism
• I expect you to be on time with cell phones
off
• Grand Rounds this Saturday!
 Grand Rounds
this Saturday, 25/9 at 9:00am
“Postpartum Hemorrhage and Maternal Mortality in
Southern Sudan”
‐featuring‐
Deng Simon Garang and Alyaa Helal,
3rd year students

Learn about physiology of hemorrhage, the physiology of pregnancy, and

physical findings related to cardiovascular instability
 Review Questions

• What are the major energy carriers for

metabolism?
• What is the rate-limiting step of glycolysis?
• What is the net ATP production from
glycolysis?
• What is the end product for anaerobic
glycolysis?
 Review Questions--Answers

• NADH, FADH2, ATP

• Step 3: phosphorylation of F6P using
PFK-1
• Net ATP production: 2
• Lactate
 Lecture objectives (1 of 2)

• What is the purpose of the TCA cycle?

Where does it occur?
• Name the steps of the TCA cycle (hint:
CIOOPOHO).
• What are the regulated steps? What
enzymes are used? What activates and
inhibits these enyzmes?
 Lecture objectives (2 of 2)

• What is the purpose of the Electron Transport

Chain? Where does it occur?
• Name the 5 Complexes. What is the pathway
of electron flow?
• What is oxidative phosphorylation? How
does it result in ATP?
• What is the net ATP production of aerobic
glycolysis?
• Name the inhibitors of the ETC. How does
uncoupling work?
 Central Equation of Glycolysis

Glucose has 3 choices:

1) Glycolysis to pyruvate
2) Storage as glycogen
3) Oxidation via Pentose
Phosphate Pathway

Glucose + 2Pi + 2ADP + 2NAD+ --> 2Pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
Alternative Fates of Pyruvate

1) Oxidative decarboxylation
to acetyl coA (next step of
aerobic glycolysis)

2) Carboxylation of pyruvate to
oxaloacetate (first step of
gluconeogenesis)

3) NOT SHOWN: Reduction of

pyruvate to ethanol (in
yeast and microorganisms,
not humans)
From Glycolysis to TCA Cycle

• Pyruvate is converted to
Acetyl CoA
• Acetyl CoA can enter the
TCA cycle
• For each pyruvate, TCA
cycle produces 2CO2, 8
electrons (accepted by
energy carriers), 1 ATP
 Tricarboxylic Acid Cycle
• Tricarboxylic acid cycle aka Krebs cycle aka
Citric Acid Cycle
• Breaks down remainder of carbon skeleton,
leaving only reducing equivalents
• Final common pathway for the aerobic
metabolism of carbohydrates, amino acids,
fatty acids--produces most ATP for animals
• Takes place in the mitochondrial matrix: it is
tied to oxidative phosphorylation (the process
by which NAD+ and FADH+ are regenerated
and ATP is produced)
Tricarboxylic Acid Cycle
 Step 1: Oxidative decarboxylation of pyruvate

• Pyruvate is shuttled to mitochondrial matrix (crosses

inner mitochondrial membrane)
• Regulated and irreversible
• Catalyzed by pyruvate dehydrogenase
 Pyruvate Dehydrogenase

• Complex of three enzymes

• Requires coenzymes such as thiamine
pyrophosphate (vitamin B1), Coenzyme A (CoA),
lipoic acid
Regulation of Pyruvate Dehydrogenase

• Acetyl CoA
directly
inhibits
• Kinase
inactivates
• Phosphatase
activates
 Acetyl CoA

Now we have acetyl CoA, a 2 carbon

molecule ready for the TCA cycle
 Quiz Yourself

• Where must pyruvate move to begin the TCA

cycle?

• What is the enzyme that catalyzes the first

step of the TCA cycle?
Overview of TCA Cycle
How to remember TCA Cycle

CIOOPOHO
Entry point:
Acetyl CoA+OAA
2C+4C = 6C
 Products of TCA Cycle

Released from cycle

2 CO2
1 GTP (like ATP)
3 NADH
1 FADH2
For every molecule of glucose, how many of
these will we get?
 Step 2: Condensation (synthesis of Citrate)

• Catalyzed by citrate synthase

• Citrate inactivates PFK-1
 Step 3: Isomerization of Citrate

• Catalyzed by aconitase
 Step 4: Oxidative Decarboxylation of Isocitrate

• 6 carbons becomes 5 carbons

• Regulated (one of the rate-limiting steps)
• Catalyzed by isocitrate dehydrogenase: activated by
ADP, Ca2+, inhibited by ATP, NADH
• Makes 1st molecule of NADH
 Step 5: Oxidative Decarboxylation of
alpha-ketoglutarate

• Regulated (one of the rate-limiting steps)

• Catalyzed by alpha-ketoglutarate dehydrogenase:
activated by ADP, inhibited by ATP, NADH
• Similar to PDH: requires CoA, TPP, lipoic acid
• Makes 2nd molecule of NADH
Step 6: Phosphorylation of Succinyl CoA

• Catalyzed by succinate CoA synthetase

• Makes 1 molecule of GTP (interchangeable
with ATP)
 Step 7: Oxidation of Succinate

• Catalyzed by succinate dehydrogenase

• Makes 1 molecule of FADH2
 Step 8: Hydration of Fumarate

• Catalyzed by fumarase
 Step 9: Oxidation of Malate

• Catalyzed by malate dehydrogenase

• Makes 3rd molecule of NADH
 Quiz Yourself

• How much NADH is produced by the TCA

cycle? How much FADH2? How much ATP?

• What happens during the condensation step

of the TCA cycle?
 Regulation of TCA Cycle

• Step 1: pyruvate to acetyl CoA (pyruvate

dehydrogenase)

• Step 4: oxidative decarboxylation of

isocitrate (isocitrate dehydrogenase)

• Step 5: oxidative decarboxylation of alpha-

ketoglutarate (alpha-ketoglutarate
dehydrogenase)
Regulation of TCA Cycle
Intro to Electron Transport Chain

We now have:
• Glycolysis: 2 ATP and 2 NADH
• TCA cycle: 2 GTP (ATP), 6 NADH, 2 FADH2
• What do we do with NADH and FADH2?

We must have a way to regenerate NAD+ and

FAD+…these are necessary for cells!
 Electron Transport Chain

• ETC receives electrons from NADH and

FADH2 (H-) and donates them to O2 to make
H2O
• Occurs in inner mitochondrial membrane
– TCA cycle was in the matrix
• The energy generated by the oxidation-
reduction reactions (removing electrons from
NADH and FADH2, donating electrons to O2)
allows oxidative phosphorylation, which
generates a lot of ATP
Inner Mitochondrial Membrane
 Composition of ETC

• Made up of 5 complexes
• Each complex accepts electrons from the complex
preceding it and donates it to the complex following it
 Pathway of electrons
• NADHComplex I (NADH dehydrogenase) 
Coenzyme Q aka ubiquinone  Complex III (cytochromes
b+c1)  cytochrome c  Complex IV (cytochrome a+a3)
O2
– NADH becomes NAD+
• FADH2Complex II (FADH2 dehydrogenase)
Coenzyme Q aka ubiquinone…O2
– FADH2 becomes FAD+
• Coenzyme Q and cytochrome c are mobile electron
carriers not part of the complexes
• Cytochrome contain heme groups (iron-sulfur ring) and
can help accept and donate electrons
Composition of ETC
 Free Energy in ETC

• As electrons are transported along the chain, free

energy is released
• The amount of free energy released determines
how many ATP can be made (from ADP)
– Phosphorylation of ADP requires 7300 cal/mol
– Transport of electrons from NADH to O2 releases
enough energy to make 3 ATP
– Transport of electrons from FADH2 to O2 releases
enough energy to make 2 ATP
 Quiz Yourself

• What are the regulated steps of the TCA cycle?

• What is the pathway of electrons when NADH is the

electron donor?

• How much ATP is made for every FADH2 that

passes electrons into the ETC?
 Accounting for ATP

We now have:
• Glycolysis: 2 ATP and 2 NADH
• TCA cycle: 2 GTP (ATP), 6 NADH, 2 FADH2
• How many ATP from 1 glucose?
• 2 + 6 + 2 +18 + 4 = 32

How do we make this ATP?

 Oxidative Phosphorylation

• Oxidative phosphorylation refers to the

synthesis of ATP using the energy created by
the electron transport chain

• Chemiosmotic hypothesis: the theory of how

the free energy of the ETC is used to produce
ATP from ADP and Pi
 Chemiosmotic Hypothesis

• Electron transport is coupled to the transport of H+

from the matrix to the intermembrane space
 Creation of Proton Gradient

• As H+ passes across the membrane, two

gradients are created: electrical and pH
ATP Synthesis

• After the protons

have been
transported to the
intermembrane
side, they reenter
the matrix
(because of
gradients) through
Complex V
• This results in the
synthesis of ATP
from ADP+Pi
Site-Specific Inhibitors of ETC

• Complex I: barbiturates
• Complex II: carboxin
• Complex III: antimycin A
• Complex IV: cyanide
• Complex V: oligomycin
Complex I: Barbiturates
Complex II: Carboxin
Complex III: Antimycin A
Complex IV: Cyanide

Cyanide
Complex V: Oligomycin
 Uncoupling Mechanism

• When the ETC is “uncoupled” from oxidative

phosphorylation, protons leak back across the
inner mitochondrial membrane into the matrix
• They do not go through ATP synthase
(Complex V)
• This means that ATP is not formed via the
proton gradient
• Example: bupivicaine
Uncoupling of ETC from Ox-Phos
 Quiz Yourself

• How much ATP do we expect to make from aerobic

glycolysis?

• What are the two gradients created by the free

energy released by the ETC?

• What is the mechanism of uncoupling?

 Lecture objectives (1 of 2)

• What is the purpose of the TCA cycle?

Where does it occur?
• Name the steps of the TCA cycle (hint:
CIOOPOHO).
• What are the regulated steps? What
enzymes are used? What activates and
inhibits these enyzmes?
 Lecture objectives (2 of 2)

• What is the purpose of the Electron Transport Chain?

Where does it occur?
• Name the 5 Complexes. What is the pathway of
electron flow?
• What is oxidative phosphorylation? How does it
result in ATP?
• What is the net ATP production of aerobic glycolysis?
• Name the inhibitors of the ETC. How does
uncoupling work?