Fat accumulation under different temperatures

PET-CT scan showing uptake and

distribution of FDG under different
temperatures.

Treatment: Two hours exposure to 19°C

(aerobic exercise helps too)

What differentiates the

Pre and Post images?
What differentiates male
and female images?
 Cold temperatures induce formation of
Brown adipose tissue (BAT)

Brown
because it has
WAT BAT more
Stores energy in large Rich in UCP1 mitochondria
lipid droplets. Provides Helps maintain body (more
insulation. temperature by cytochromes).
thermogenesis.
DOI: 10.1088/2057-1976/2/2/025005 3
 Uncoupling proteins and thermogenesis

Signaling cascade UCP1 allows the leak of Energy from oxidation

Fatty acids activate
leads to release of fatty protons dissipating the of fuels is released as
UCP1 in mitochondria
acids from lipid droplets proton gradient heat

https://doi.org/10.1016/j.biochi.2016.12.016 4
 Why do uncouplers generate heat?
• Processes that are not 100% efficient
generate heat as by product.
• The ETC and ATP synthesis are not
perfectly coupled. Not all the protons What
pumped by the ETC are used to substrates?
How does that
synthesize ATP. Proton leaks is energy lead to weight
lost as heat. loss?

5
 Why do uncouplers generate heat?
• Processes that are not 100% efficient
generate heat as by product.
• The ETC and ATP synthesis are not
perfectly coupled. Not all the protons What
pumped by the ETC are used to substrates?
How does that
synthesize ATP. Proton leaks is energy lead to weight
lost as heat. loss?
• In the presence of an uncoupler, to get
the same amount of ATP made,
mitochondrial respiration will have to run
at a faster rate.
• Faster combustion of substrates.

6
 How do protons get back into the matrix?

Option 1: Through ATP

synthase.
High membrane potential 
High ATP synthesis

Option 2: Through
uncouplers (heat generated
as side-effect).
Lower membrane potential
 Reduced ATP synthesis

https://www-nature-com.myaccess.library.utoronto.ca/articles/nrn1767 7
ATP synthesis
Chapter 21

02/14/24 8
Objectives
Describe the Describe how Identify the Distinguish the
structure and the proton-motive mechanisms factors that affect
mechanisms of force is converted required to the yield of ATP.
the F0 and F1 into ATP by ATP transport NADH
subunits. synthase. and ADP into the
mt.

9
 If you could be any enzyme from BIOC13,
what enzyme would you be? and why?

Proton
gradient and “I would be ATP
ATP synthesis
synthase because it
shows how positivity can
Cashing H+ for drive productivity :) ”
ATP
B.T.
“All enzymes are beautiful, but the ATP synthase is one of
Shuttles the most beautiful as well as one of the most unusual and
across important. […] Its importance is illustrated by the estimate
membranes that an active graduate student synthesizes more than
his or her body weight of ATP in a day.”

Paul D. Boyer

10
 Enzymatic mechanism for ATP synthesis
Failed to identify the phosphorylated
intermediate in ATP synthesis.

Elucidated the “binding change

mechanism” for ATP synthesis

“I had had no previous courses or

research experience in biochemistry and
was uncertain about my career choice.
By the end of my first year of graduate
study the fascination of biochemical
understanding and the addictive effect of
experimental attempts to uncover new
knowledge had firmly launched me
toward a career in biochemical
research.”

https://www.jbc.org/article/S0021-9258(20)55940-1/fulltext 11
 Hands-on experience with ATP synthase

https://sketchfab.com/3d-models/atp-
https://pinshape.com/items/33
synthase-rotation-
854-3d-printed-atp-synthase
f9ad55a805d443389dd8ae5c80351fb1

12
 ATP structure
How it started How it is going

Next to the discovery of

DNA, the discovery that
ATP synthesis is powered
by a proton gradient is one
of the two major
advancements in Biology in
this century.
13
 How to elucidate a protein structure?
Method 2

Method 1

F1 : soluble, amenable to x-ray crystallography

F0: too flexible, required electron cryo-microscopy
(cryo-EM)

https://www.nature.com/news/the-revolution-will-not-be-crystallized-a-new-method-sweeps-through-structural-biology-1.18335 14
 Starting simple: structure elucidation from a simple
organism

Bacterial ATP synthases

are the simplest forms of
the enzyme.

eLife 2019;8:e43128 DOI: 10.7554/ELIFE.43128 15

 Bacterial ATP synthase
Bacillus PS3 is a thermophilic bacteria.
E. coli can be genetically manipulated.
Expressed thermoresistant Bacillus PS3
ATP synthase in genetically tractable E. coli
for cryo-EM imaging.

Cryo-EM allows high-resolution structure

determination of biomolecules in solution
without crystallization.
(2017 Nobel Prize in Chemistry).

16
 ATP synthase structure elucidation
To determine a structure by
cryoelectron microscopy,
researchers scatter many copies of
the molecule on a surface, and then
combine images of thousands of
them to build up a coherent
structure.

In some cases, the molecules adopt

several different shapes, so by
classifying these images correctly,
researchers can observe different
conformations of the molecule.
Spot the differences

https://pdb101.rcsb.org/motm/72 17
 Bacterial ATP synthase

Cryo-EM allows building atomic model.

Representative 2D ATP synthases.

18
 ATP synthase subunits
(Positive side)
Two components:
F0 subunit embedded in the inner mt
membrane contains the proton channel
Made by subunits a, b and c

F1 subunit contains the catalytic activity of

the synthase.
Made by subunits a, b, g, d, e
β3: Active sites, each subunit has different
interactions with γ subunit.
Matrix
γ and ε form the stalk

a alpha, b beta, g gamma, d delta, e epsilon 19

 ATP synthase has two motors
(Positive side)
F0 (transmembrane)
Electric motor (turns) c a
Powered by flow of H+ across membrane

g e (Negative side)
Rotor or Connects the motors
When F0 turns, F1 turns too
b
F1 (matrix)
Chemical motor
The F0 motor forces
the F1 motor to turn

d
20
 Structure of the proton channel F0

3 subunits: a, b, c
c subunits Or Glu
Ring comprised of 12 identical
c chains (blue, # of subunits
can change from 8-15
depending on species)

Asp or Glu found in the middle

of the helices (- charged).
a subunit
Arg found in the a subunit
Matrix (+charged).
Asp and Glu can lose
protons becoming Connected to F1 through
negatively charged central γ and ε, and exterior
(ideal when you need to column (b in orange)
move protons)
21
Structure of the catalytic unit F1

5 subunits: 3a, 3b, 1g,1d, 1e

Uses the power of rotational

motion to build ATP.

Three steps:
1. Trapping of ADP and Pi
2. Formation of new
phosphate bond
3. Release of ATP and
recharge

22
 ATP synthase forms dimers

Mitochondrial ATP synthases

form dimers.
Association stabilizes individual
enzymes to the rotational forces
required for catalysis, and
promotes curvature of
membrane.

DOI: 10.1042/BST20110773
23
 ATP synthase mechanism
(as viewed (as viewed
from the from the
High [H+] top) matrix)
(as viewed
from the
top)

(as viewed ATP synthase uses a proton motive force

from the as a source of energy to drive a
Low [H+] matrix) mechanical rotary mechanism that leads
to the chemical synthesis of ATP from ADP
and Pi.
 ATP synthase nucleotide binding sites

Look at the position of the g Loose

subunit. Open
Each of the b subunits is distinct
because each interacts with a
different face of the g subunit. b a
b
a
g a

(as viewed
g is the “knob” that rotates
“squeezing” the b subunits.
What b subunit has more space,
b
from the less space?
bottom) Tight

25
ATP synthase nucleotide binding sites are not equivalent
The binding change mechanism accounts for the
synthesis of ATP in response to proton flow.

The three catalytic β subunits of the F1 component can

exist in three conformations:

In the L (loose) form, nucleotides are trapped in

the β subunit. Binds ADP + Pi.

In the T (tight) form, ATP is synthesized from ADP

and Pi.

In the O (open) form, nucleotides can bind to or

be released from the β subunit.

Rotation of the g subunit drives the interconversion of

the three forms.
26
 Rotation of gamma subunit (120⁰ CCW)

Tight Tight Open

Open Loose Loose
(as viewed
from the
bottom)

Tight Open Loose

Rotation of the gamma subunit causes sequential In what order do b subunits

conformational changes in the three beta subunits change conformation?
that alter their substrate-binding abilities. a) L  T  O
b) O  T  L
c) T  L  O
d) T  O  L 27
 Formation of ATP with rotation of gamma subunit

Loose Tight Open

ADP+
ADP + Pi  Tight ATP
Open Pi Loose
ATP

Open Loose
Tight

ATP in the O-form site can now

ADP and Pi in the L-form trap the ADP and Pi bound in the T-form
depart from the enzyme to be
substrates. combine to form ATP.
replaced by ADP and Pi

28
Rotation of the γ subunit interconverts the β subunits

Form ATP Open Release Recharge

What b No two subunits are ever in the same conformation.

subunit POV is Each subunit cycles through the three conformations.
this figure
representing? Hexamer does not move, g rotates in 120 ⁰ increments.
Green Each 120⁰ rotation releases 1 ATP.
Yellow
blue
29
 Proton flow across inner mitochondrial membrane

Subunit a, which abuts the subunits c ring, has

two channels that reach halfway into the a
subunit. One half channel opens to the
Subunits c
intermembrane space and the other to the matrix.
(transmembrane
helices)
The movement of protons through the half-
channels from the high proton concentration of
Half-channel  Subunit a the inner membrane space to the low proton
(two half concentration of the matrix powers the rotation of
channels) the c ring.

Glutamate or aspartate are found in the middle of

Half-channel
the c subunit.
If glutamate is charged (unprotonated)  c
subunit does not move into hydrophobic interior of
membrane.

30
Rotation drives work

H+

H+

31
 Protonation of amino acid drives rotation

H+

H+
Glutamic
acid

Glutamate (or aspartate) will receive

a proton from the intermembrane
Glutamate space and become glutamic acid
(aspartic acid).
Glutamic acid (and aspartic acid)
H+ can lose one proton to form
glutamate (aspartate).
32
Proton flow drives rotation of c ring

1 2 3

6 5 4

Unprotonated Glu, no rotation 33

 Proton flow drives rotation of c ring

1 2 3
1. Unprotonated subunit a.
2. Proton enters the half-channel of the
subunit a from the positive side of the
membrane.
3. Proton that entered subunit a is loaded
into one c subunit.
4. Proton binds to a glutamate or
aspartate residue on one of the subunits
of the c ring. 6 5 4
5. Glutamic or aspartic acid in the other
half-channel releases proton inside the
matrix.
6. Rotation brings the deprotonated subunit
to the position where it can be loaded
with another proton from the
intermembrane space.

Unprotonated Glu, no rotation 34

 Assembling F0 + F1 together
As the c ring rotates, the g and e subunits turn inside the hexamer of the F1 subunit.
Rotation of the g subunit promotes synthesis of ATP (axle).
The exterior column formed by the b chains and d subunit anchor the hexamer to prevent it from
moving (stator).

proton
ADP/ATP

35
 Proton
gradient and
ATP synthesis

NADH
H+ ATP
Cashing H+ for
ATP
FADH2

Shuttles What is the ratio of H+/ATP?

across
membranes

36
ATP yield from glucose to CO2 and H2O
One rotation = 10 protons One rotation = 3 ATP

10 protons = 3.3 protons per

3 ATPs ATP
~3 protons per ATP
(to be continued after shuttles)

The number of c rings determines Each 360 degree rotation of the g

the number of protons required to subunit leads to synthesis and
synthesize 1 molecule of ATP. release of 3 ATP.

37
 What is the ratio of H+/ATP? It depends …

Which one
is more
efficient?
The ____ c
subunits,
the ____
protons are
required to
rotate 360⁰

Electron density map from crystals of Oligomers of chloroplast ATP

yeast mitochondrial ATP synthase synthase visualized using atomic
shows a ring of 10 c subunits force microscopy shows a ring of
14 c subunits
38
Stoichoimetry of H+/ATP is not fixed, it varies with system

The number of c rings determines the number of protons required to synthesize a molecule
of ATP.

Yeast 10 c subunits
Each molecule of ATP generated requires the transport of 10/3= 3.3 protons per ATP.

Vertebrates 8 c subunits
8/3= 2.7 protons per ATP.
Making vertebrate ATP synthase the most efficient known.

On average
~3 protons per ATP

39
 ATP currency exchange ratio: From ETC to ATP
4H+ 4H+ 2H+
Number of H+ translocated across membrane by ETC
If NADH  10 H+
If FADH2  6 H +
Electron Transport Chain

NADH
H+ ATP

FADH2
ATP synthase

~3 protons per ATP

40
 ATP currency exchange ratio: From oxidation of NADH and
FADH2 to ATP

Number of H+ translocated across membrane by ETC

If NADH  10 H+
If FADH2  6 H +

10 for NADH or 6 for FADH2

+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐫𝐚𝐧𝐬𝐥𝐨𝐜𝐚𝐭𝐞𝐝 𝐚𝐜𝐫𝐨𝐬𝐬 𝐢𝐧𝐧𝐞𝐫 𝐦𝐭 𝐦𝐞𝐦𝐛𝐫𝐚𝐧𝐞 𝐚𝐟𝐭𝐞𝐫 𝐨𝐱𝐢𝐝𝐚𝐭𝐢𝐨𝐧 𝐛𝐲 𝐄𝐓𝐂
+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐡𝐚𝐭 𝐦𝐮𝐬𝐭 𝐟𝐥𝐨𝐰 𝐭𝐡𝐫𝐨𝐮𝐠𝐡 𝐭𝐡𝐞 𝐀𝐓𝐏 𝐬𝐲𝐧𝐭𝐡𝐚𝐬𝐞 𝐭𝐨 𝐠𝐞𝐧𝐞𝐫𝐚𝐭𝐞 𝐞𝐚𝐜𝐡 𝐀𝐓𝐏
~3 protons per ATP

10
= 𝐴𝑇𝑃𝑠 𝑝𝑒𝑟 𝑁𝐴𝐷𝐻
3? ? ?
10 H + for 3 ATPs, or 3 H + for 1 ATP
But … (to be continued after shuttles)
6
= 𝐴𝑇𝑃𝑠 𝑝𝑒𝑟 𝐹𝐴𝐷𝐻2
3? ? ?
 Where metabolites are made matters
Number of protons Number of protons Number of ATPs
translocated into required to make F0 do produced by F1 after one
intermembrane space by one full rotation full rotation
ETC

NADH
FADH2 H+ ATP

Number of H+ translocated across membrane by ETC But that is assuming that NADH and FADH2
If NADH  10 H+ are already inside the mitochondrial matrix
If FADH2  6 H +

42
 This NADH
is outside
the mt

Proton
gradient and ADP and Pi
ATP synthesis need to get
inside mt

Cashing H+ for
ATP

This NADH
Shuttles is inside the
across mt
membranes

43
 Location, location, location

What ATP synthase needs

to make ATP:
1. Protons from NADH Find the
and FADH2 pumped “sneaky”
into intermembrane proton
space
2. ADP in the matrix
3. Phosphate (Pi) in the
matrix

(Positive side)

https://doi.org/10.1016/j.bbamcr.2016.03.015 44
 Location, location, location

What ATP synthase needs

to make ATP:
1. Protons from NADH
and FADH2 pumped
into intermembrane
1. Shuttles to move cytoplasmic NADH
space
A. Glycerol-3-phosphate shuttle
2. ADP in the matrix B. Malate-aspartate shuttle
3. Phosphate (Pi) in the
matrix 2. Shuttles to move ADP
3. Shuttles to move Pi

45
1. Shuttles allow movement of protons across membranes

Problem: One of the functions of the respiratory Matrix

chain was to regenerate NAD+ for use in glycolysis.
How is cytosolic NADH reoxidized to NAD+ under
A. Glycerol
aerobic conditions? 3-Pi shuttle

Further complication: Inner mt membrane is

impermeable to NADH and NAD+

B. Malate-
Solution: Instead of carrying NADH across aspartate
membranes, transfer electrons from NADH through shuttle
shuttles.

46
 1.A. Glycerol 3-phosphate shuttle
In muscle and brain, electrons from
cytoplasmic NADH can enter the
electron- transport chain by using the
glycerol phosphate shuttle.

The electrons are transferred from

How is this NADH to FADH2 and subsequently
shuttle
going to to Q to form QH2.
affect the
potential
yield of
ATP?
Loss of energy
6 H+ per
NADH
from
cytosol
47
 Deja Vu

Glycerol 3-Pi Glycerol 3-Pi

dehydrogenase dehydrogenase
(cytoplasmic) (mitochondrial)

48
 1.B. Malate-aspartate shuttle
Cytoplasmic Malate
dehydrogenase
In heart, kidney and liver, electrons from
NADH cytoplasmic NADH are used to generate
NAD+ mitochondrial NADH in malate-aspartate shuttle.

The malate-aspartate shuttle consists of two

membrane antiporters and four enzymes.

No loss of energy

10 H+ per
Mitochondrial Malate
NADH
dehydrogenase from
cytosol
50
2. ADP entry coupled to ATP exit by ATP-ADP translocase

The translocase catalyzes

the coupled entry of ADP into
the matrix and the exit of ATP
from it.

The binding of ADP (1) from

the cytoplasm favors
eversion of the transporter (2)
to release ADP into the
matrix (3). Subsequent
binding of ATP from the
matrix to the everted form (4)
favors eversion back to the
original conformation (5),
releasing ATP into the
cytoplasm (6).

51
3. Mitochondrial transporters for ions and charged molecules

H+ H+ H+ H+
H+ H+ H+
H+ H+ For each ATP,
a phosphate is
required.
Getting the
phosphate in,
will require an
extra H+ inside
the matrix.

An OH- out is
equivalent to a
H+ in
To get the phosphate in, a OH- will be exported. This neutralizes one H+, reducing the
proton gradient.
Adds 1 H to the cost of 1 ATP (because it transports 1 OH- out).
+

52
 Recap: substrate oxidation steps
Per Glucose

Glycolysis
2 PDH 2 Citric acid
• 6 NADH
• 2
NADH reaction
NADH cycle
FADH2

cytoplasm mitochondria

• 1 NADH =
Electron
10 H+ Protons
transport
• 1 FADH2 to ATP?
chain
= 6 H+

53
 ATP currency exchange ratio: From oxidation of NADH and
FADH2 to ATP

Number of H+ translocated across membrane by ETC

If NADH  10 H+
If FADH2  6 H +

+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐫𝐚𝐧𝐬𝐥𝐨𝐜𝐚𝐭𝐞𝐝 𝐚𝐜𝐫𝐨𝐬𝐬 𝐢𝐧𝐧𝐞𝐫 𝐦𝐭 𝐦𝐞𝐦𝐛𝐫𝐚𝐧𝐞 𝐚𝐟𝐭𝐞𝐫 𝐨𝐱𝐢𝐝𝐚𝐭𝐢𝐨𝐧 𝐛𝐲 𝐄𝐓𝐂
+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐡𝐚𝐭 𝐦𝐮𝐬𝐭 𝐟𝐥𝐨𝐰 𝐭𝐡𝐫𝐨𝐮𝐠𝐡 𝐭𝐡𝐞 𝐀𝐓𝐏 𝐬𝐲𝐧𝐭𝐡𝐚𝐬𝐞 𝐭𝐨 𝐠𝐞𝐧𝐞𝐫𝐚𝐭𝐞 𝐞𝐚𝐜𝐡 𝐀𝐓𝐏
ATP currency exchange ratio: From oxidation of NADH and
FADH2 to ATP

+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐫𝐚𝐧𝐬𝐥𝐨𝐜𝐚𝐭𝐞𝐝 𝐚𝐜𝐫𝐨𝐬𝐬 𝐢𝐧𝐧𝐞𝐫 𝐦𝐭 𝐦𝐞𝐦𝐛𝐫𝐚𝐧𝐞 𝐚𝐟𝐭𝐞𝐫 𝐨𝐱𝐢𝐝𝐚𝐭𝐢𝐨𝐧 𝐛𝐲 𝐄𝐓𝐂
+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐡𝐚𝐭 𝐦𝐮𝐬𝐭 𝐟𝐥𝐨𝐰 𝐭𝐡𝐫𝐨𝐮𝐠𝐡 𝐭𝐡𝐞 𝐀𝐓𝐏 𝐬𝐲𝐧𝐭𝐡𝐚𝐬𝐞 𝐭𝐨 𝐠𝐞𝐧𝐞𝐫𝐚𝐭𝐞 𝐞𝐚𝐜𝐡 𝐀𝐓𝐏

10 H + for 3 ATPs, or 3 H + for 1 ATP

But … need extra H + to translocate Pi
Adjusted
10+3=13 H+ for 3 ATPs or 4 H + for 1 ATP
ATP currency exchange ratio: From oxidation of NADH and
FADH2 to ATP

+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐫𝐚𝐧𝐬𝐥𝐨𝐜𝐚𝐭𝐞𝐝 𝐚𝐜𝐫𝐨𝐬𝐬 𝐢𝐧𝐧𝐞𝐫 𝐦𝐭 𝐦𝐞𝐦𝐛𝐫𝐚𝐧𝐞 𝐚𝐟𝐭𝐞𝐫 𝐨𝐱𝐢𝐝𝐚𝐭𝐢𝐨𝐧 𝐛𝐲 𝐄𝐓𝐂
+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐡𝐚𝐭 𝐦𝐮𝐬𝐭 𝐟𝐥𝐨𝐰 𝐭𝐡𝐫𝐨𝐮𝐠𝐡 𝐭𝐡𝐞 𝐀𝐓𝐏 𝐬𝐲𝐧𝐭𝐡𝐚𝐬𝐞 𝐭𝐨 𝐠𝐞𝐧𝐞𝐫𝐚𝐭𝐞 𝐞𝐚𝐜𝐡 𝐀𝐓𝐏

10
= 2.5 𝐴𝑇𝑃𝑠 𝑝𝑒𝑟 𝑁𝐴𝐷𝐻
4
10 H + for 3 ATPs, or 3 H + for 1 ATP
But … need extra H + to translocate Pi
Adjusted 6
= 1.5 𝐴𝑇𝑃𝑠 𝑝𝑒𝑟 𝐹𝐴𝐷𝐻2
10+3=13 H+ for 3 ATPs or 4 H + for 1 ATP 4
 ATP currency exchange ratio: From oxidation of NADH and
FADH2 to ATP

Number of H+ translocated across membrane by ETC

If NADH  10 H+
If FADH2  6 H +

+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐫𝐚𝐧𝐬𝐥𝐨𝐜𝐚𝐭𝐞𝐝 𝐚𝐜𝐫𝐨𝐬𝐬 𝐢𝐧𝐧𝐞𝐫 𝐦𝐭 𝐦𝐞𝐦𝐛𝐫𝐚𝐧𝐞 𝐚𝐟𝐭𝐞𝐫 𝐨𝐱𝐢𝐝𝐚𝐭𝐢𝐨𝐧 𝐛𝐲 𝐄𝐓𝐂
+
𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐇 𝐭𝐡𝐚𝐭 𝐦𝐮𝐬𝐭 𝐟𝐥𝐨𝐰 𝐭𝐡𝐫𝐨𝐮𝐠𝐡 𝐭𝐡𝐞 𝐀𝐓𝐏 𝐬𝐲𝐧𝐭𝐡𝐚𝐬𝐞 𝐭𝐨 𝐠𝐞𝐧𝐞𝐫𝐚𝐭𝐞 𝐞𝐚𝐜𝐡 𝐀𝐓𝐏

10
= 2.5 𝐴𝑇𝑃𝑠 𝑝𝑒𝑟 𝑁𝐴𝐷𝐻
4
10 H + for 3 ATPs, or 3 H + for 1 ATP
But … need extra H + to translocate Pi
Adjusted 6
= 1.5 𝐴𝑇𝑃𝑠 𝑝𝑒𝑟 𝐹𝐴𝐷𝐻2
10+3=13 H+ for 3 ATPs or 4 H + for 1 ATP 4
ATP yield

3 H+ drive synthesis 1 ATP

(plus 1 H+ to transport ADP and Pi into matrix)
4 H+ drive synthesis 1 ATP

Yield of NADH
10/4 = 2.5 molecules of cytoplasmic ATP from NADH.
Yield of FADH2
6/4= 1.5 molecules of cytoplasmic ATP from FADH2.

NADH  2.5 ATP

FADH2  1.5 ATP

58
Energy yield from oxidative metabolism

59
Energy yield from oxidative metabolism

60
 Energy yield from oxidative metabolism
NADH made inside
mitochondria → no need to
move reducing equivalents
across membrane

Variables that affect

yield:
Cells using the glycerol 3-
phosphate shuttle to move • Shuttle used to
NADH from cytosol → electrons move NADH
enter the respiratory chain as into
FADH2, yield is 1.5 ATP mitochondria
• Substrate used

Prokaryotes and cells using the

malate/aspartate shuttle → no
energy cost to move reducing How many
equivalents across membrane
ATPs in the
liver?

61
NADH and FADH2 are oxidized when ATP is made

The synthesis of ATP from

ADP and Pi controls the flow of
electrons from NADH and
FADH2 to oxygen.

The availability of NAD+ and

FAD in turn control the rate of
the citric acid cycle (CAC).

When ADP concentration rises

(Ex: active muscle that is
consuming ATP), the rate of
oxidative phosphorylation
increases to meet the ATP
needs of the cell.

62
63
 Mitochondrial diseases have disparate phenotypes yet
common mechanisms
Roles of mitochondria:
- Aerobic energy production,
- Reactive oxygen species formation

The brain (central nervous system) is

particularly vulnerable to mitochondria
dysfunction.
Why? 20% of the body’s metabolic
demand, and neurons depend on
oxidative phosphorylation for functioning

Neurological disorders implicated with

defects in mitochondrial dysfunction:
Alzheimer’s disease, Autism, Epilepsy
Lou Gehrig’s
DOI 10.15252/emmm.201505079 64
 Activity of complex I and V are reduced in autistic
individuals

What is the
conclusion of
the figure?

Scattered plots of activities of mitochondrial ETC complexes (I, II, III, IV and V) in the
frontal cortex of brain from autism and control subjects. *P<0.05.

https://www-nature-com.myaccess.library.utoronto.ca/articles/tp201368 65
 Prevalence of mt disorder in ASD population is higher than in
general population

Mt dysfunction results in less energy available to

fuel the high-energy needs of the brain and
muscles, and leaves free radicals in the system
where they can cause damage.

Not all Autism Spectrum Disorders (ASD) involve

mitochondrial disease. But, mt dysfunction has
been found repeatedly in this group of children and
adults.

This association opens the door to potential

treatments (changes in diet or supplements).

https://doi.org/10.1016/j.spen.2020.100829 66
 Samson and Shintay

Grizzlies do not actually

hibernate but enter a
“winter sleep”.
• Body T⁰ drops slightly.
• Metabolic activity is
very low.
• Do not eat or drink.
• Lose body fat every
day.
How to they warm up
when it is time to wake-
up?

67
 Uncoupling proteins and thermogenesis

• UCP1 or thermogenin is used by animals to control

thermogenesis.
• Cell-specific (brown adipose tissue) expression of the
UCP1 protein leads to heat production under aerobic
conditions.
• During final weeks of hibernation, norepinephrine
levels increase and activate UCP1 expression at the
base of the neck, which warms the blood traveling to
the brain and signals the end of a long winter nap.
• Allows protons to cross the inner mt membrane and
thereby uncouple electron transport from ATP
synthesis.

68
Coming
up

69