A Diet to Die For: An Exploration of Oxidative Phosphorylation

by
Terry Platt, Department of Biology, University of Rochester
Eric Ribbens, Department of Biological Sciences, Western Illinois University
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 145pound weight category? Well, its here!
Whats here?
The solution! DNP!
Just a Few Little Pills
Cheryl picked up the invoice. Seventy-one bucks for 11 tablets? What is musleman.com anyhow?
Charles nodded. Its not cheap, but its 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?
The Plan
I dont know, and I dont care. But it should help me get rid of these eight pounds
in only a week. Its fast, and its 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, Its my secret for now; besides, he wouldnt
care he just wants us to win, and this is my first college match!
Goes Awry
Two days later, Cheryl met her brother crossing campus after dinner and learned
that he had taken an extra two pills in the afternoon, since he thought he wasnt
losing weight fast enough to make his cutoff next weekend.

You know, you dont look so good you seem kind of flushed, and youre
breathing pretty fast have you been running?
Actually, Charles confided, I havent, and Im a little scared because my
body feels like its racing, even though Im not
You Can Lose More Than Weight
I feel kind of nauseous, too, and weak in the knees, continued Charles, and Im
sweating like a horse look how damp my shirt is.
We better get you to Student Health, urged Cheryl. The sooner the better.
Well, okay, but dont tell my coach about this, or he might not let me
compete on Saturday!
Fortunately, Student Health saw him quickly. They immediately called an
ambulance for emergency treatment, as they recognized that there was more than
Charles weight at stake.
Two Days Later
A scared Cheryl and her parents listened nervously to the emergency room doctor.
You said he was taking DNP, 2,4-dinitrophenol? Well, youve all had a very
serious scare, but it looks like hes going to be okay. Well keep him here for a few
days, just to be certain.
Cheryl sniffed. Hell be mad to miss his match, and I dont understand! It
was working so well. And it was for sale over the internet! What happened?
Dr. Adams frowned.
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!
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 1930s

to stimulate metabolism and promote weight loss. Concernsled 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.

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.

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?

CQ#1: Choose the description below that best completes the statement
oxidative phosphorylation is the process in mitochondria by which:
A. 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.
Ox Phos, as its often called, is carried out in your mitochondria, began Dr.
Adams.
Cheryl interrupted, Oh, yeah, I remember - theyre 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 thats only the first half, the oxidative part, of the story.
What About Phosphorylation?
This is the part I have trouble with, said Cheryl, because what I dont get is
how converting oxygen into water helps you make ATP.
Dr. Adams replied, Well, youre in good company. For two decades or
more, most scientists studying this process also didnt get it.
So lets 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).
Thats the second half.
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.
CQ#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?

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.

The answer is A.
Cyanide inhibits Complex IV, known as cytochrome oxidase,
preventing the transfer of electrons to molecular oxygen.
Conclusion: Cyanide prevents oxygen consumption, and because ATP
production is also prevented, ATP synthesis must require electron transport.
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
CQ#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. 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.
The answer is A.
Blocking ATP synthase also prevents the consumption of oxygen.
Conclusion: Not only does ATP synthesis require electron transport, but
electron transport requires ATP synthesis.
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.
How Does Coupling Work?

For many years, this phenomenon was enigmatic; it wasnt 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!

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 lets look at a picture!
ATP synthase:

What About the Protons?

Thats pretty neat, but where do the protons come from to make it work?
Cheryl wanted to know.Ah, youve 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.
Three Linked Events
Remember what you know about the inner membrane of mitochondria?

1. Electron transport occurs laterally between complexes embedded in the lipid

bilayer, ending with the reduction of molecular oxygen to water.
2. In concert with this electron transport, protons are pumped out of the
mitochondria into the inter-membrane space by Complexes I, III and IV,
creating a higher pH (less acidic) within the matrix.
3. 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.
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. Lets look at a picture youve seen before.
Peter Mitchells Chemiosmotic Hypothesis
Protons are pumped out (by the ETC) and flow back in
(via ATP synthase) to make ATP.

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?
CQ#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.
The answer is B: DNP acts as an uncoupler!
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?

What Does DNP Actually Do?

Heres 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?

Phyllic or Phobic?
Cheryl responded, Hydrophobic like greasy stuff, right? Ive watched my
dad use benzene to get the grease off his hands when hes been working on
the car, and I know that it doesnt 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 heres an application to think about
CQ#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 (samplle 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.
The correct answer is C, Dr. Adams lectured.
And heres 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 ion species (H +
and Cl-) cannot cross lipid bilayer structures.
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 wouldnt 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 youre
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?
What Happens to 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.
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, its 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?

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!

For Warm Babies and Bears

Heat generation by non-shivering thermo-genesis 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.

Epilogue A Look Back in Time

In the 1950s and 60s, 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.
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. outside (lower Ph, 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.

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?

CQ#6. You isolate intact mitochondria and equilibrate them in a buffered

solution at pH 9, containing 0.1 M KCl and ADP plus P i, 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 K +
ionophore). 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. 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.
Start with equilibrated mitochondria

Mitochondria are placed in pH 9 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.
What Did This Experiment Prove

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
Mitchells proposal.

Behind Mitchells Novel Proposal

Peter Mitchells insights into DNPs 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.