1
A DIET TO DIE FOR:
AN EXPLORATION OF
OXIDATIVE PHOSPHORYLATION
Based on a PowerPoint by
Terry Platt, Department of Biology, University of Rochester
Eric Ribbens, Department of Biological Sciences, Western Illinois University
2
The “Energy Burner”
“Cheryl! Come here!” Charles called to his twin sister
excitedly.
“What?” asked Cheryl.
“You know how my JV wrestling coach wants me to
get down to the 145-pound weight category? Well, it’s
here!”
“What’s here?”
“The solution! DNP!”
3
Just a Few Little Pills
Cheryl picked up the invoice. “Seventy-one bucks for
11 tablets? What is musle-man.com anyhow?”
Charles nodded. “It’s not cheap, but it’s been
scientifically proven to work. Here are the
instructions. Take one pill the first day, two tablets
each of the next four days…”
“So this really works? What does it do?”
4
The Plan…
“I don’t know, and I don’t care. But it should help
me get rid of these eight pounds in only a week. It’s
fast, and it’s proven to work.”
Cheryl was intrigued. “Maybe this could help me
trim back too. I could sure use it in a few crucial
places… Hey, does your coach know about this?
Maybe it could help the whole team!”
“Heck, no!” Charles exclaimed, “It’s my secret for
now; besides, he wouldn’t care – he just wants us
to win, and this is my first high school match!”
5
…Goes Awry
Two days later, Cheryl talked to her brother after
dinner and learned that he had taken an extra two
pills in the afternoon, since he thought he wasn’t
losing weight fast enough to make his cutoff next
weekend.
“You know, you don’t look so good – you seem kind
of flushed, and you’re breathing pretty fast – have
you been running?”
“Actually,” Charles confided, “I haven’t, and I’m a little
scared because my body feels like its racing, even
though I’m not…”
6
You Can Lose More Than Weight
“I feel kind of nauseous, too, and weak in the knees,”
continued Charles, “and I’m sweating like a horse – look
how damp my shirt is.”
“We better get you to the Doctor’s Office,” urged Cheryl.
“The sooner the better.”
“Well, okay, but don’t tell my coach about this, or he
might not let me compete on Saturday!”
Fortunately, Charles’s physician agreed to see him
quickly. They immediately called an ambulance for
emergency treatment, as they recognized that there
was more than Charles’ weight at stake.
7
Two Hours Later …
A scared Cheryl and her parents listened nervously to
the emergency room doctor, Dr. Adams.
“You said he was taking DNP, 2,4-dinitrophenol? Well,
you’ve all had a very serious scare, but it looks like he’s
going to be okay. We’ll keep him here for a few days,
just to be certain.”
Cheryl sniffed. “He’ll be mad to miss
his match, and I don’t understand! It
was working so well. And it was for
sale over the Internet! What
happened?”
Dr. Adams frowned.
8
A Diet You May Die For
“Medically, DNP is one of those drugs
where the ‘therapeutic dose,’ which is
the amount that will produce desired
results in half of those who take it, is only
a little lower than the ‘lethal dose’ (LD50),
where half of those who take it die. For
drugs to be medically approved, the LD50
must be much higher than the
therapeutic dose.”
“Oh my gosh,” Cheryl said to her parents
in a whisper. “Those two extra little pills
could have killed my brother!”
9
Charles is Alive and Stable
While Charles was recovering, Cheryl found a paper “Dying
to be thin: A dinitrophenol related fatality.”* The authors note
that 2,4 dinitrophenol:
• “was originally used as an explosive and later introduced in the 1930’s
to stimulate metabolism and promote weight loss. Concerns…led to
DNP being banned as a dietary aid in 1938.”
• A 22-yr-old male patient arrived at the ER 16 hr after his last dose of
DNP with a temperature of 102 degrees Fahrenheit and sweating
profusely.
• He became agitated and delirious, was mechanically cooled and given
IV drug treatment to counter the DNP, but within another hour his heart
slowed, stopped, and, despite resuscitative efforts, he died.
• “Advertisements claim DNP safe at the dose our patient ingested. It is
widely available and with the potential to cause severe toxicity is an
understudied public health concern.”
*McFee et al. (2004) Vet. Hum. Toxicol. 46: 251-254.
10
Why Does DNP Do This?
• Cheryl read some more. In an article titled
“Weight loss and 2,4-dinitrophenol poisoning,”*
• a 27-yr-old female admitted to the ER “complaining of fatigue,
nausea, and excessive sweating” had begun taking new diet
tablets the week before, and had “doubled the recommended dose
for faster results.” Despite heroic efforts to save her, she died 7
hours after admission.
• the authors stated that “DNP causes a hyper-metabolic state by
uncoupling oxidative phosphorylation. Energy is released in the
mitochondria as heat… Toxic doses will result in uncontrolled
thermogenesis leading to hyperthermia and systemic responses to
elevated body temperature.”
*Tewari et al. (2009) Brit. J. Anaesthesia 102: 566-567.
11
What is Oxidative Phosphorylation?
• Cheryl had only a vague recollection of this process,
recalling that it had something to do with metabolic
breakdown of energy rich compounds, electrons,
phosphorylation, and the role of ATP.
• What do you remember, and what do you think?
12
Question #1: Choose the description below that
best completes the statement :
Oxidative phosphorylation is the process in
mitochondria by which…
Electrons reduce O2 to H2O and this causes
ATP to be made.
B. Glycolysis produces more ATP than is
needed to activate its pathway.
C. Oxygen is used to cause phosphorylation of
biological molecules.
D. Synthesis of ATP is dependent on the
passage of electrons through the electron
transport chain.
A.
13
The answer is D.
Without electrons passing through the electron transport chain, there
is no generation of an electrochemical gradient (or “proton pressure”).
14
Here’s a Quick Review
“Ox Phos, as it’s often called, is carried out
in your mitochondria…,” began Dr. Adams.
Cheryl interrupted, “Oh, yeah, I remember they’re ‘the powerhouses of the cell’!”
“...where much of your cellular energy, in
the form of ATP, is made. The majority of
electrons derived from oxidation [remember
OILRIG?] of nutrients (sugars, fats,
proteins) in your body end up passing
through the electron transport chain (ETC)
that is embedded in the mitochondrial inner
membrane. They combine in the end with
molecular oxygen to produce water. But
that’s only the first half, the ‘oxidative’ part,
of the story.”
15
What About Phosphorylation?
“This is the part I have trouble with,” said
Cheryl, “because what I don’t get is how
converting oxygen into water helps you
make ATP.”
Dr. Adams replied, “Well, you’re in good company.
For two decades or more, most scientists studying
this process also didn’t get it.
“So let’s just look for a moment at the relationship
between the ETC and the activity of the enzyme,
ATP synthase, that makes ATP in mitochondria by
“phosphorylating” ADP with inorganic phosphate
(usually termed Pi). That’s the second half.”
16
Mitochondrial Experiments
• Scientists have isolated mitochondria from the cells
they normally occupy, and they retain many of their
functions, including the ability to carry out “Ox Phos.”
• All they require is some substrate that could be
oxidized (succinate works well), ADP + Pi (as
precursors), and appropriate buffers and salts.
• ETC activity can be detected by the consumption of
oxygen (using an oxygen electrode).
• ATP synthesis can be measured by the appearance of
labeled ATP from radioactive ADP precursor.
17
Some Questions
“Let’s see if you have the basics, Cheryl,” Dr.
Adams proposed.
“First, I’m sure you know that cyanide is a nasty
poison – that’s because it blocks Component IV
(cytochrome oxidase) of the ETC.”
“What do you think will happen if you add cyanide
to a test tube with mitochondria that are actively
carrying out Ox Phos?”
18
Question #2: You incubate isolated intact mitochondria in a
buffered solution containing succinate (an oxidizable
substrate) and ADP plus Pi. Upon adding cyanide (an inhibitor
of Complex IV, cyto-chrome oxidase), you examine the effect
on oxygen consumption and the production of ATP. What do
you predict?
A.
B.
C.
D.
Oxygen will not be consumed, and no ATP will be
produced.
Oxygen will be consumed, but no ATP will be
produced.
Oxygen will not be consumed, but ATP will be
produced.
Oxygen will be consumed, and ATP will be
produced.
19
Cyanide
added
Succinate
added
ADP + Pi
added
ATP synthesized
Oxygen consumed
The answer is A.
Cyanide inhibits Complex
IV, known as cytochrome
oxidase, preventing the
transfer of electrons to
molecular oxygen.
Time
Conclusion: Cyanide prevents oxygen consumption,
and because ATP production is also prevented, ATP
synthesis must require electron transport.
20
Another Experiment
“Now, in a similar situation, instead of using cyanide, you
add an inhibitor of ATP synthase, such as the antibiotic
oligomycin.
Think carefully, because the answer to this may be a little
tricky…”
21
Question #3: You incubate isolated intact mitochondria in a
buffered solution with succinate (an oxidizable substrate)
and ADP plus Pi. Upon adding oligomycin, an antibiotic
inhibitor of ATP synthase, you examine the effect on oxygen
consumption and the production of ATP. What do you
predict?
A.
B.
C.
D.
Oxygen will not be consumed, and no ATP will be
produced.
Oxygen will be consumed, but no ATP will be
produced.
Oxygen will not be consumed, but ATP will be
produced.
Oxygen will be consumed, and ATP will be produced.
22
Oligomycin
added
Succinate
added
ADP + Pi
added
ATP synthesized
Blocking ATP
synthase also
prevents the
consumption of
oxygen. How
can this be?
Oxygen consumed
The answer is A.
Time
Conclusion: Not only does ATP synthesis require electron
transport, but electron transport requires ATP synthesis.
23
The Concept of “Coupling”
Dr. Adams continued, “The obvious part is that
because ATP synthase is blocked, no ATP can be
synthesized.
The challenging question is: how does this prevent
the consumption of oxygen, since none of the
components of the Electron Transport Chain have
been directly affected?
Scientists have decided that this unexpected result
should be called ‘coupling’ between electron
transport and ATP synthesis.”
24
How Does Coupling Work?
• For many years, this phenomenon
was enigmatic; it wasn’t until a British
scientist named Peter Mitchell came
up with his unusual “Chemiosmotic
Hypothesis” that things began to
make sense.
• In parallel, it was becoming clearer
how the enzyme ATP synthase
worked – as a molecular motor!
Peter Mitchell
(1920-1992)
25
Motors in Cells?
Dr. Adams smiled. “Elegant enzyme experiments have
shown that this enzyme is a true molecular motor, if you
can believe that – driven by the flow of protons through a
channel, which
causes physical
rotation of a…
…but let’s look at
a picture!”
ATP synthase:
26
What About the Protons?
• “That’s pretty neat, but where do the protons come from to
•
•
•
•
make it work?” Cheryl wanted to know.
“Ah, you’ve hit on the key question that helps understand
coupling, and in the process reveals how DNP works! But,
one step at a time.
As examination of the ETC progressed, data indicated that
protons were being pumped out of the mitochondria in
concert with the passage of electrons down the ETC.
Many scientists argued that this proton pumping was only a
tangential byproduct of electron transport, and unrelated to
its main function.
Peter Mitchell, however, suspected a direct involvement,
and many of his experiments began to support it.”
27
Three Linked Events
Remember what you know about the inner
membrane of mitochondria?
1.
2.
3.
Electron transport occurs laterally between
complexes embedded in the lipid bilayer, ending
with the reduction of molecular oxygen to water.
In concert with this electron transport, protons are
pumped out of the mitochondria into the intermembrane space by Complexes I, III and IV,
creating a higher pH (less acidic) within the matrix.
ATP synthase (Complex V) is a molecular motor
also in the lipid bilayer, driven by proton passage
through a channel that lets them back in again.
28
It Starts to Make Sense
Dr. Adams continued: “With the realization
that ATP synthase required the passage of
protons, driven by a proton gradient (which
Mitchell called his ‘Proton Motive Force’),
where they could flow ‘downhill,’ the puzzle
became resolved….
Before that, it was believed that….well, never
mind,” explained Dr. Adams. “Let’s look at a
picture you’ve seen before.”
29
Peter Mitchell’s Chemiosmotic Hypothesis
Protons are pumped out (by the ETC) and flow back in
(via ATP synthase) to make ATP.
30
What Happens with DNP?
“With this in mind, remember when you read that DNP was
an ‘uncoupler’ of Ox Phos? What would that mean in
terms of the ETC and ATP synthesis?” asked Dr. Adams.
Cheryl thought, and came up with an idea.
What idea do you have for this question?
31
Question #4: You incubate isolated intact mitochondria in a
buffered solution with succinate (an oxidizable substrate) and
ADP plus Pi. You add oligomycin (the ATP synthase inhibitor),
then the compound DNP (2,4-dinitrophenol), and examine the
effect on oxygen consumption and the production of ATP.
What do you predict? Hint: What happened when people took
this as a weight-loss drug?
A.
Oxygen will not be consumed, and no ATP will be produced.
B.
Oxygen will be consumed, but no ATP will be produced.
C.
Oxygen will not be consumed, but ATP will be produced.
D.
Oxygen will be consumed, and ATP will be produced.
32
Oligomycin
added
Succinate
added
ADP + Pi
added
DNP
added
ATP synthesized
Oxygen consumed
The answer is B: DNP acts as an uncoupler!
Time
Recall: Electron transport requires ATP synthesis except in the presence of DNP, where oxygen continues
to be consumed, though no ATP is being made. The
mitochondria are said to be uncoupled. How is this?
33
What Does DNP Actually Do?
“Here’s the structure of DNP, with a ring that looks sort of
like benzene – do you think it would be more soluble in
water, or in a hydrophobic solvent?”
34
Phyllic or Phobic?
Cheryl responded, “Hydrophobic – like greasy stuff,
right? I’ve watched my dad use benzene to get the
grease off his hands when he’s been working on the
car, and I know that it doesn’t mix with water.”
“Yes, good thinking.” Dr. Adams circled the –OH group
on his diagram. “But, unlike benzene, you can see that
DNP is a ‘weak acid’ due to its –OH group, which just
means that some of the time it can shed its proton,
becoming –O- + H+. This is unlike HCl, which is a
strong acid, and thus completely dissociates in water.
So here’s an application to think about…”
35
Question #5: You have created some artificial
membrane vesicles (spherical lipid-bilayer enclosed
droplets) that have a higher pH inside than the aqueous
solution outside. To sample 1 of these vesicles you add
a little HCl, and to the other (sample 2) you add some
DNP (with equivalent acidity) and then measure the
internal pH of the vesicles. What do you predict?
A. Internal pH will be lowered for both 1 and 2.
B. Internal pH will be unchanged for both 1 and 2.
C. Internal pH will be unchanged for 1 but lowered for 2.
D. Internal pH will be lowered for 1 but unchanged for 2.
36
“The correct answer is C,” Dr. Adams lectured.
“And here’s why: when protonated, that is, carrying its H+
proton as you saw in the figure, DNP will be not only
uncharged, but very hydrophobic. It is hence readily
soluble in lipid bilayers such as those enclosing these
vesicles (and those of the mitochondrial inner membrane).
Thus DNP can readily carry protons across the membrane
from a higher concentration (lower pH) outside, and
release them to the lower concentration (higher pH) inside.
This equilibrates the pH inside and outside.
HCl cannot do this, because it is not hydrophobic, and its
charged ions (H+ and Cl-) cannot cross lipid bilayer
structures.”
37
What Happens With a Leak?
“That is really cool,” Cheryl blurted out, “and if we think of
mitochondria as just sort of glorified vesicles, then DNP
would make their inside pH the same as the outside pH –
the difference would be gone! But wouldn’t that be a big
problem, because now the Electron Transport Chain would
have to keep working like crazy with its proton pumping just
to keep up? Sort of like a bicycle tire with a leak, when
you’re trying to fill it?”
“Exactly right!” Dr. Adams exclaimed, “And what do you think
that ‘working like crazy’ might need a lot of, and generate a
lot of…?”
38
What Happens to the Wasted Energy?
“A lot of oxygen!” Cheryl said, “Because to pump
all those protons, oxygen has to be there to accept
the electrons being transported along at the same
time.”
“And heat!” cried Dr. Adams. “Does this fit with the
effects you know that DNP has on people?”
Cheryl thought. “Yes: rapid breathing because they
need oxygen, sweating from the increase in body
temperature due to the heat generated, and
fatigue because ATP stops being made, even
though lots of calories are being burned.”
39
To Lose Weight,
You May Lose Your Life…
• Dr. Adams summarized, “So you now realize why DNP
is so dangerous. Although it will cause weight loss, it’s
within a very narrow dosage window, and small
physiological differences between individuals may shift
its effects into the lethal range. The ‘If one pill is good,
two pills must be better’ approach has a deadly flaw
with this drug. Your brother is actually a very lucky
young man.
• But Cheryl, did you know that some organisms co-opt
this concept and use it in a well-controlled fashion?”
40
A Controlled Energy Burn!
• Human babies, bears, and some rodents have
“brown fat” - specialized fat cells, much richer
than normal in mitochondria (which is why they
are brown).
• The inner membranes of these mitochondria
have a protein called thermogenin.
• It provides a proton channel from outside to
inside, dissipating the proton gradient without
making ATP, thus causing the ETC to run faster,
creating...heat!
41
For Warm Babies and Bears
H+
H+
H+
H+
H+
X
Heat
H+
Thermogenin
(uncoupling
protein)
H+
H+
• Heat generation by
“non-shivering thermogenesis” keeps human
babies (who have a
high surface to volume
ratio) warm when they
need to be.
• Thus organisms have
evolved a mechanism
to control what DNP
does in an uncontrolled
way, and to exploit it in
a helpful way.
42
Epilogue – A Look Back in Time
In the 1950’s and 60’s, the mechanism for how energy
derived from the transport of electrons was connected to
the generation of ATP was regarded as mysterious.
• Many eminent scientists believed that there had to be a
transient high energy phosphoryl chemical intermediate,
which was nicknamed X~P.
• This intermediate was predicted to capture the energy
released during electron transport, and then transfer it to
ADP, forming ATP – a “Chemical Coupling” model.
• When Peter Mitchell proposed the “Chemiosmotic
Hypothesis,” it was highly controversial.
43
Alternative Possibilities
• The Chemiosmotic Hypothesis seemed to fit
some observations that could not be accounted
for by Chemical Coupling, including:
• The higher pH inside vs. lower pH outside (thus more
protons) of the mitochondrial matrix.
• The known pumping of protons outward, regarded by
many as being peripheral to oxidative phosphorylation
• Nonetheless, Mitchell had many serious and
vocal critics who seemed to resent any challenge
to the Chemical Coupling model.
44
Hypothesis Testing
• The Chemical Coupling model predicted that ATP
synthesis could not occur in the absence of
electron transport, because X~P was only formed
by the action of the ETC.
• The Chemiosmotic Hypothesis predicted that the
driving force for ATP synthesis comes from the
proton gradient (or “Proton Motive Force” as
Mitchell termed it) directly.
• Eventually, experiments like the following one
were performed. What do you think will happen?
45
Question #6. You isolate intact mitochondria and
equilibrate them in a buffered solution at pH 9, containing
0.1 M KCl and ADP plus Pi, but without succinate. You
collect them by centrifugation, and quickly resuspend them
in a new buffer at pH 7, without KCl , but with valinomycin
(a molecule that increases membrane permeability for K+
ions). Note: the K+ rushing out will create a huge positive
charge differential. What do you predict will be the result
on oxygen consumption and the production of ATP?
A.
B.
C.
D.
Oxygen will not be consumed, and no ATP will be produced.
Oxygen will be consumed, but no ATP will be produced.
Oxygen will not be consumed, but ATP will be produced.
Oxygen will be consumed, and ATP will be produced.
46
Start with equilibrated mitochondria
• Mitochondria are placed in pH 9
[K+] = [Cl-] = 0.1 M
H+ = 10-9 M
H+ = 10-9 M
[K+] = [Cl-] = 0.1 M
H+
H+ = 10-9 M
Intermembrane
space
buffer and 0.1 M KCl, and
allowed to reach equilibrium.
The proton (10-9 M) and the
potassium (0.1 M)
concentrations are thus
identical in the inter-membrane
space and the matrix.
These mitochondria are now
shifted into pH 7 buffer, no KCl,
with valinomycin present.
47
Can an Artificial Proton Gradient Drive ATP Synthesis?
Intermembrane
space H+
H+
H+
H+
H+ = 10-7 M
Cl-
Cl-
ClH+
ClCl-
K+
H+ = 10-9 M
K+
[K+] < [Cl-]
valinomycin
Cl-
Now, DpH = 2, and K+
flows out, giving a
charge imbalance
(negative inside) that
helps pull in protons.
The answer is C. ATP is
synthesized, in the
complete absence of any
electron transport or
oxygen consumption.
48
What Did This Experiment Demonstrate?
• ATP synthesis correlated with the formation of an
electrochemical gradient of protons, and
• In the complete absence of any electron transport!
• This result supported the Chemiosmotic Hypothesis
developed by Peter Mitchell, and ruled out those that
require ATP synthesis to be solely dependent on
electron transport.
• Many scientific opponents vigorously challenged it as
being heretical if not impossible, and it took them
another decade or more to be convinced by Mitchell’s
proposal.
49
Behind Mitchell’s Novel Proposal
Peter Mitchell’s insights into DNP’s function as an uncoupler
led him, in part, to set aside traditional thinking and to
propose his controversial alternative, the “Chemiosmotic
Hypothesis.”
Throughout his studies, he kept an open mind, and was
always trying to test between alternative hypotheses rather
than trying to “prove” a favorite one. This was in striking
contrast to many of those who either did not understand or
could not accept his ideas.
His ideas revolutionized the way in which we think about
oxidative phosphorylation, photosynthesis, and energy
transduction in living cells.
50
A Brilliant but Modest Researcher
In 1978, Peter Mitchell flew to Stockholm to accept his Nobel
Prize and offered a gracious comment to the scientific
community in his acceptance speech:
"I find most remarkable and admirable...the
altruism and generosity with which former
opponents of the chemiosmotic hypothesis
have not only come to accept it, but have
actively promoted it to the status of a theory."