Last edited: 8/26/2021

8. ELECTRON TRANSPORT CHAIN (PART 3)

ETC: ATP Synthesis, Interfering Agents, and Energetics Medical Editor: Jona Frondoso

OUTLINE

I) ATP SYNTHASE
II) ATP SYNTHESIS
III) AGENTS THAT INTERFERE WITH OXIDATIVE
PHOSPHORYLATION
IV) ENERGETICS
V) APPENDIX
VI) REVIEW QUESTIONS
VII) REFRENCES

I) ATP SYNTHASE Figure 1. The Fo subunit and the F1 subunit of the ATP
synthase (University of Cambridge, 2015)
Also known as Complex V
Multi-subunit enzyme of the inner mitochondrial membrane
(2) F0 SUBUNIT (Figure 1)
that catalyzes the formation of ATP from ADP and Pi,
accompanied by the flow of protons from the o denoting oligomycin-sensitive (University of Cambridge, 2015)
intermembrane space into the mitochondrial matrix Pore found in the inner membrane portion
An F-type ATPase (Nelson & Cox, 2008) o Proton pore – through which protons leak as fast as
Its parts can be divided into: they are pumped by electron transfer
o 2 parts linked by the peripheral and central stalks Composed of single copies of each of the ff. (Figure 2):
 F1 catalytic domain
 Fo domain (i) ATP6 or a
o 4 parts (ii) b
 Rotor
(iii) c
 Stator
 Rod (iv) e
 Knob (v) f
(vi) g
(A) 2 PARTS OF ATP SYNTHASE (vii) ATP8 or AL6 subunits
(1) F1 SUBUNIT (Figure 1) Mammalian enzymes contain additional supernumerary
subunits j and k (University of Cambridge, 2015)
• Peripheral membrane protein
• Composed of a stalk and a head piece
• First factor recognized as essential for oxidative
phosphorylation (University of Cambridge, 2015)
• Globular assembly of five different proteins with the
stoichiometry 3:3:1:1:1 (Figure 2) (University of Cambridge, 2015)

(i) Alpha
3 alpha subunits - arranged alternately around a
central alpha-helical coiled coil in the gamma
subunit

(ii) Beta Figure 2. Structure of the ATP synthase and its supernumerary
subunits (University of Cambridge, 2015)
3 alpha subunits - arranged alternately around a
central alpha-helical coiled coil in the gamma (B) 5 PARTS OF ATP SYNTHASE
subunit
Passage of protons through a pathway between the c-ring
Has a pocket containing ADP and inorganic
and subunit a releases energy to drive the clockwise
phosphate (Pi)
rotation of the rotor (as viewed from the membrane) during
(iii) Gamma ATP synthesis (Figure 3)

1 gamma subunit – protrudes from α3β3 (1) Rotor

subcomplex Within the membrane
Spins clockwise when protons flow past it down the proton
(iv) Delta
gradient
1 delta – associated with the gamma subunit’s
foot (2) Stator
Anchored in the membrane
(v) Epsilon Holds the knob stationary
1 epsilon – associated with the gamma subunit’s
(3) Rod
foot
Or stalk
Extending into the knob
Also spins, activating catalytic sites in the knob

ELECTRON TRANSPORT CHAIN (PART 3) METABOLISM: Note #8. 1 of 5

(4) Knob
Stationary
Has three catalytic sites
Joins inorganic phosphate to ADP to make ATP
Figure 3. The rotor, stator, knob, and rod of the ATP synthase
(Mathews, van Holde, Appling, & Anthony-Cahill, 2013)
II) ATP SYNTHESIS
(A) PROTON GRADIENT
ETC is coupled to the phosphorylation of ADP by the
transport (“pumping”) of protons (H+) across the inner
mitochondrial membrane from the matrix to the IMS (Harvey
and Ferrier, 2011)
Proton gradient between the intermembrane space and the
mitochondrial matrix is later on established
o Mitochondrial matrix [H+] = 10-9 mM
 Lesser proton concentration
o IMS: [H+] = 10-7 mM
 Greater proton concentration
Proton gradient established moves from high (IMS) to low
(mitochondrial matrix)
Protons make the IMS positively charged → depolarizing
the membrane → activates the ATP synthase
Proton-gradient drives the release of ATP from the enzyme
surface
(B) CHEMIOSMOSIS
Chemiosmosis – movement of protons from high
concentration to low concentration through the ATP
synthase (Figure 4)
The a subunit has one pore opening to IMS and the other
pore opening to the mitochondrial matrix
Protons run down through the pores of a subunit from high
concentration to low concentration
o A passive transport (does not require ATP)
o Arginine-210 coordinates the process
o Arginine-210 takes the proton coming through the pore
facing the IMS and switches it over to put it into the
pore facing the mitochondrial matrix
o Once Arginine-210 puts the proton into the other pore,
it swings back causing the rotor of the ATP synthase
to rotate.
Figure 4. Chemiosmosis in ATP synthesis
2 of 5
(C) PROTON-MOTIVE FORCE
Rotor and rod rotates against the stator generating a
proton-motive force
As the protons move from high concentration to low
concentration, the ATP synthase absorbs some potential
energy.
ATP synthase uses the potential energy from the rotation
to fuse the ADP and Pi in the pocket of beta catalytic
subunit forming ATP (Figure 4)
(D) OXIDATIVE PHOSPHORYLATION
Process in which ATP is formed as a result of the transfer
of electrons from NADH or FADH 2 to O2 (Figure 4)
Generates much larger amounts of energy than substrate-
phosphorylation
III) AGENTS THAT INTERFERE WITH OXIDATIVE
PHOSPHORYLATION
(A) UNCOUPLING OF PHOSPHORYLATION OF
ELECTRON TRANSFER
(1) 2,4- DNP
2,4-dinitrophenol
Creates a hydrophobic pore in the inner mitochondrial
membrane through which protons can run down from the
IMS to the mitochondrial matrix (an alternative route for
H+ to move through)
o Hydrophobic pore – cannot synthesize ATP
Uncouples ATP synthase from the electron transport chain
→ ↓ ATP production and ↑ ADP
↑ ADP → ↑ glycolytic, transition step, Krebs cycle activity
→ ↑ metabolic rate but ↓ ATP → ↑ thermogenicity (heat
production) → beneficial to weight loss (Figure 5)
Historical use
o For weight loss
o Extremely dangerous diet pill
Figure 5. Uncoupling of ATP synthase from the electron
transport chain by 2,4 DNP and thermogenin
(2) Brown adipose tissue
Major site of thermogenesis in newborn
Brown color indicates the abundance of mitochondria
Not present in adults
Produces the chemical thermogenin
o Thermogenin – creates a pore in the inner
mitochondrial membrane similar to 2,4-DNP
Presence of proton-conducting pore → provides an
alternative route for protons to move through → uncouples
ATP synthase form the ETC → ↓ ATP production and ↑
ADP
↑ ADP → ↑ glycolytic, transition step, Krebs cycle activity
→ ↑ metabolic rate but ↓ ATP → ↑ thermogenicity (heat
production) (Figure 5)
METABOLISM: Note #8. ELECTRON TRANSPORT CHAIN (PART 3)
 (B) INHIBITION OF ELECTRON TRANSFER
(1) Cyanide
Inhibits cytochrome oxidase (Complex IV)
o Cytochrome oxidase: transported electrons in its
high-energy state Fe2+, ½ O2, and free two protons (H+)
are brought together, reducing O2 to water
Cyanide poisoning → inhibits Complex IV → inhibits
transfer of electrons to ½ O2 → histoxic hypoxia (Figure 6)
o Histoxic hypoxia
 Refers to the reduction in ATP production by the
mitochondria due to a defect in the cellular usage
of oxygen
 There are plentiful amounts of oxygen that can be
utilized
Figure 7. Direct inhibition of ATP synthase by oligomycin and
aurovertin

Shown in Table 2 is a summary of the different agents that

interfere with oxidative phosphorylation

IV) ENERGETICS

(1) NADH
Pumps out one proton at Complex I, one proton at
Complex III, and one more proton at Complex IV
1 proton that comes down = 1 ATP
Hence, 1 NADH can generate 3 ATP
Figure 6. Inhibition of cytochrome oxidase by cyanide and (2) FADH2
carbon monoxide
Pumps out one proton at Complex III and one proton at
(2) Carbon monoxide (CO) Complex IV
Also inhibits cytochrome oxidase (Complex IV) 1 proton that comes down = 1 ATP
CO poisoning → inhibits Complex IV → inhibits transfer of Hence 1 FADH2 can generate 2 ATP
electrons to ½ O2 → histoxic hypoxia (Figure 6)
Can also binds to iron-containing heme group in Table 1. Tally of End-Products of Glycolysis, Transition State,
and Krebs Cycle and ATP Generated
hemoglobin
ATP Final
Direct
Process generated ATP
Product
(3) Antimycin A per molecule
Blocks electron transfer from cytochrome b to cytochrome 2 ATP x1 2a
c1 Glycolysis
2 NADH x3 6b
Historical use:
o Antibiotic Transition State 2 NADH x3 6b
o Today, it is commonly used in research to study the
effects of the inhibition of cellular respiration 6 NADH x3 18 b
o x2 4b
Krebs Cycle 2 FADH2
(C) INHIBITION OF ATP SYNTHASE X1 2a
2 ATP
Direct inhibition of ATP formation
Total yield per 38 c
(1) Aurovertin glucose
a
ATP generated via substrate-level phosphorylation
Inhibits F1 subunit of the ATP synthase b
ATP generated via oxidative phosphorylation
Inhibited F1 subunit → failure to synthesis ATP (Figure 7) c
Total ATP generated from both substrate-level and
(2) Oligomycin oxidative phosphorylation
Inhibits Fo subunit of the ATP synthase
Inhibited Fo subunit → failure to synthesis ATP (Figure 7)

ELECTRON TRANSPORT CHAIN (PART 3) METABOLISM: Note #8. 3 of 5

 V) APPENDIX

Figure 8. Overview of the ATP synthesis, its points of interference, and the ETC tally.

Table 2. Agents That Interfere with Oxidative Phosphorylation

Type of Interference Compound Target / Mode of Action
Cyanide Inhibition cytochrome oxidase
Carbon monoxide
Antimycin A Blocks electron transfer from cytochrome b to cytochrome c1
Inhibition of electron transfer Myxothiazol Prevent electron transfer from FE-S center to ubiquinone
Rotenone
Amytal
Piericidin A
DCMU Competes with Qb for binding site in PSII
Aurovertin Inhibits F1
Inhibition of ATP synthase Oligomycin Inhibits Fo and CFo
Venturicidin
DCCD Blocks proton flow through Fo and CFo
FCCP Hydrophobic proton carriers
DNP
Uncoupling of phosphorylation
Valinomycin K+ ionophore
from electron transfer
In brown fat, forms proton-conducting pores in inner
Thermogenin
mitochondrial membrane
Inhibition of ATP-ADP exchange Atractyloside Inhibits adenine nucleotide translocase

* DCMU is 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCCD, dicyclohexylcarbodiimide; FCCP, cyanide-p-

trifluoromethoxyphenylhydrazone; DNP, 2,4-dinitrophenol

4 of 5 METABOLISM: Note #8. ELECTRON TRANSPORT CHAIN (PART 3)

 VI) REVIEW QUESTIONS

Which subunit of the ATP synthase contains the ADP

and inorganic phosphate?
a. Alpha
b. Beta
c. Delta
d. Gamma

Arrange the following statements sequentially.

1. Depolarization of the membrane
2. Creation of a proton gradient
3. Chemiosmosis
4. Activation of the ATP synthase
5. Rotation of the rotor rod against the stator
6. Fusion of ADP and inorganic phosphate
7. ATP synthase absorbs potential energy

a. 2-1-3-4-7-5-6
b. 2-1-4-3-5-7-6
c. 4-2-1-3-7-5-6
d. 4-1-2-3-5-7-6

Which pf the following directly inhibits the ATP

synthase?
a. Oligomycin
b. Cyanide
c. DNP
d. Atractyloside

Which of the following statements regarding ETC

energetics is correct?
a. 3 ATP is generated from 1 FADH2
b. 2 ATP is generated from 1 NADH
c. The total ATP yield per glucose is 38.
d. Most of the ATP are generated via substrate-level
phosphorylation.

Which of the following is not true about the

mechanism of brown fat in relation to heat
production?
a. It produces thermogenin
b. It uncouples ATP synthase from ETC
c. It forms proton conducting pores.
d. It produces a chemical that directly inhibits the ATP
synthase

CHECK YOUR ANSWERS

VII) REFRENCES
● Harvey, R., & Ferrier, D. (2011). Lippincott's Illustrated Reviews
- Biochemistry (5th ed.). Philadelphia: Lippincott Williams & WIlkins.
● Mathews, C., van Holde, K., Appling, D., & Anthony-Cahill, S.
(2013). Biochemistry (4th ed.). USA: Pearson.
● Nelson, D., & Cox, M. (2008). Lehninger Principles of
Biochemistry (4th ed.). New York: W.H. Freeman.
● University of Cambridge. (2015). Subunit Composition of ATP
Synthasae. Retrieved from MRC: Mitochondrial Biology Unit:
http://www.mrc-mbu.cam.ac.uk/projects/2679/subunit-composition

ELECTRON TRANSPORT CHAIN (PART 3) METABOLISM: Note #8. 5 of 5