The transfer of electrons down the electron transport chain creates a proton gradient across the inner mitochondrial membrane, but this alone does not produce ATP. The chemiosmotic hypothesis explains how ATP is synthesized using this proton gradient. Specifically, as protons are pumped across the membrane at complexes I, III, and IV, a gradient forms. ATP synthase then uses the potential energy of protons flowing back through the membrane to phosphorylate ADP, producing ATP. Inhibitors like oligomycin block ATP synthase and prevent ATP production, tightly coupling electron transport to phosphorylation.

1. Oxidative Phophorylation

2. The transfer of electrons down the electron transport chain is
energetically favored because NADH is a strong electron donor and
molecular oxygen is an avid electron acceptor. However, the flow of
electrons from NADH to oxygen does not directly result in ATP synthesis.
A) Chemiosmotic hypothesis
The chemiosmotic hypothesis (also known as the Mitchell hypothesis)
explains how the free energy generated by the transport of electrons
by the electron transport chain is used to produce ATP from ADP + Pi.
1. Proton pump:
Electron transport is coupled to the phosphorylation of ADP by the
transport (“pumping”) of protons (H+) across the inner
mitochondrial membrane from the matrix to the inter membrane
space at Complexes I, III, and IV. This process creates an electrical
gradient (with more positive charges on the outside of the
membrane than on the inside) and a pH gradient (the outside of
the membrane is at a lower pH than the inside. The energy
generated by this proton gradient is sufficient to drive ATPirectly
result in ATP synthesis.

3. synthesis. Thus, the proton gradient serves as the common
intermediate that couples oxidation to phosphorylation.
2. ATP synthase:
The enzyme complex ATP synthase (Complex V) synthesizes ATP
using the energy of the proton gradient generated by the
electron transport chain. [Note: It is also called F1/Fo ATPase
because the isolated enzyme can catalyze the hydrolysis of
ATP to ADP and Pi.] The chemiosmotic hypothesis proposes
that after protons have been pumped to the cytosolic side of
the inner mitochondrial membrane, they reenter the matrix
by passing through a channel in the membrane-spanning
domain (Fo ) of Complex V, driving rotation of Fo and, at the
same time, dissipating the pH and electrical gradients. Fo
rotation causes conformational changes in the extra-
membranous F1 domain that allow it to bind ADP + Pi,
phosphorylate ADP to ATP, and release ATP.

4. a. Oligomycin:
This drug binds to the Fo (hence the letter o) domain of ATP
synthase, closing the H+ channel, preventing reentry of
protons into the mitochondrial matrix, and thus preventing
phosphorylation of ADP to ATP. Because the pH and
electrical gradients cannot be dissipated in the presence of
this drug, electron transport stops because of the difficulty
of pumping any more protons against the steep gradients.
This dependency of cellular respiration on the ability to
phosphorylate ADP to ATP is known as respiratory control,
and is the consequence of the tight coupling of these
processes. Electron transport and phosphorylation are,
therefore, again shown to be tightly coupled processes.
Inhibition of one process inhibits the other.
[Note: Respiratory control also results from decreased
availability of ADP or Pi.]

5. Uncoupling proteins (UCP):
UCPs occur in the inner mitochondrial membrane of mammals,
including humans. These carrier proteins create a “proton leak,”
that is, they allow protons to re-enter the mitochondrial matrix
without energy being captured as ATP. The energy is released as
heat, and the process is called nonshivering thermogenesis. UCP1,
also called thermogenin, is responsible for the heat production in
the brown adipocytes of mammals. UCP1 is activated by fatty acids.
Brown fat, unlike the more abundant white fat, uses almost 90% of
its respiratory energy for thermogenesis in response to cold in the
neonate, and during arousal in hibernating animals. However,
humans appear to have little brown fat (except in the newborn),
and UCP1 does not appear to play a major role in energy balance.
[Note: Other uncoupling proteins (UCP2,
UCP3) have been found in humans, but their significance
remains unclear.]

6. c. Synthetic uncouplers:
Electron transport and phosphorylation can also be
uncoupled by compounds that increase the
permeability of the inner mitochondrial membrane
to protons. The classic example is 2,4-dinitrophenol,
a lipophilic proton carrier that readily diffuses
through the mitochondrial membrane. This
uncoupler causes electron transport to proceed at a
rapid rate without establishing a proton gradient,
much as do the UCPs. Again, energy is released as
heat rather than being used to synthesize ATP. In high
doses, aspirin and other salicylates uncouple
oxidative phos -phorylation. This explains the fever
that accompanies toxic overdoses of these drugs.

7. B. Membrane transport systems
The inner mitochondrial membrane is impermeable to most
charged or hydrophilic substances. However, it contains
numerous transport proteins that permit passage of specific
molecules from the cytosol (or more correctly, the
intermembrane space) to the mitochondrial matrix.
1. ATP-ADP transport:
The inner mitochondrial membrane requires specialized carriers
to transport ADP and Pi from the cytosol(where ATP is used
and converted to ADP in many energyrequiring reactions) into
mitochondria, where ATP can be resynthesized. An adenine
nucleotide carrier imports one molecule of ADP from the
cytosol into mitochondria, while exporting one ATP from the
matrix back into the cytosol.
[Note: A phosphate carrier is responsible for transporting Pi from
the cytosol into mitochondria.]

8. 2. Transport of reducing equivalents:
The inner mitochondrial membrane lacks an NADH
transporter, and NADH produced in the cytosol cannot
directly enter the mitochondrial matrix. However, two
electrons of NADH (also called reducing equivalents)
are transported from the cytosol into the matrix using
substrate shuttles. In the glycerol phosphate shuttle
two electrons are transferred from NADH to
dihydroxyacetone phosphate by cytosolic glycero
phosphate dehydrogenase. The glycerol 3-phosphate
produced is oxidized by the mitochondrial isozyme as
FAD is reduced to FADH2. CoQ of the electron transport
chain oxidizes FADH2

9. The glycero phosphate shuttle, therefore, results in the
synthesis of two ATPs for each cytosolic NADH
oxidized. This contrasts with the malate-aspartate
shuttle which produces NADH (rather than FADH2) in
the mitochondrial matrix and, therefore, yields three
ATPs for each cytosolic NADH oxidized by malate
dehydrogenase as oxaloacetate is reduced to malate.
A transport protein carries malate into the matrix.

10. C) Inherited defects in oxidative
phosphorylation
Thirteen of the approximately 120 polypeptides required for oxidative
phosphorylation are coded for by mtDNA and synthesized in mitochondria,
whereas the remaining mitochondrial proteins are synthesized in the
cytosol and transported into mitochondria. Defects in oxidative
phosphorylation are more likely a result of alterations in mtDNA, which has
a mutation rate about ten times greater than that of nuclear DNA. Tissues
with the greatest ATP requirement (for example, central nervous system,
skeletal and heart muscle, kidney, and liver) are most affected by defects in
oxidative phosphorylation. Mutations in mtDNA are responsible for several
diseases, including some cases of mitochondrial myopathies and Leber
hereditary optic neuropathy, a disease in which bilateral loss of central
vision occurs as a result of neuroretinal degeneration, including damage to
the optic nerve. The mtDNA is maternally inherited because mitochondria
from the sperm cell do not enter the fertilized egg.

11. D) Mitochondria and apoptosis
The process of apoptosis or programmed cell death may
be initiated through the intrinsic (mitochondrial-
mediated) pathway by the formation of pores in the
outer mitochondrial membrane. These pores allow
cytochrome c to leave the intermembrane space and
enter the cytosol. Once in the cytosol, cytochrome c, in
association with proapoptotic factors, activates a
family of proteolytic enzymes (the caspases), causing
cleavage of key proteins and resulting in the
morphologic and biochemical changes characteristic of
apoptosis.

12. Substrate shuttle for transport of electrons
Glycerol 3-phosphate shuttle Malate aspartate shuttle