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A Diet to Die For: An Exploration of Oxidative Phosphorylation

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1 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

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 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 college match!”

5 …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 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 Student Health,” 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, 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.

7 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, 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:

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:

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 CQ#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. Glycolysis produces more ATP than is needed to activate its pathway. Oxygen is used to cause phosphorylation of biological molecules. Synthesis of ATP is dependent on the passage of electrons through the electron transport chain.

13 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.”

14 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.”

15 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.

16 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?”

17 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.

18 The answer is A. Cyanide added Cyanide inhibits Complex IV, known as cytochrome oxidase, preventing the transfer of electrons to molecular oxygen. Succinate added Oxygen consumed ATP synthesized ADP + Pi added Time Conclusion: Cyanide prevents oxygen consumption, and because ATP production is also prevented, ATP synthesis must require electron transport. 18

19 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…”

20 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? 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.

21 The answer is A. Blocking ATP synthase also prevents the consumption of oxygen. How can this be? Oligomycin added Succinate added Oxygen consumed ATP synthesized ADP + Pi added Time Conclusion: Not only does ATP synthesis require electron transport, but electron transport requires ATP synthesis.

22 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.”

23 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!

24 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:

25 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.”

26 Three Linked Events Remember what you know about the inner membrane of mitochondria? 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 inter-membrane 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.

27 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.”

28 Peter Mitchell’s Chemiosmotic Hypothesis
Protons are pumped out (by the ETC) and flow back in (via ATP synthase) to make ATP. 28

29 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?

30 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? 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.

31 The answer is B: DNP acts as an uncoupler!
Oligomycin added DNP added Oxygen consumed Succinate added ATP synthesized ADP + Pi added Uncoupled 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? 31

32 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?”

33 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…”

34 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.

35 “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 ion species (H+ and Cl-) cannot cross lipid bilayer structures.”

36 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…?”

37 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.”

38 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?”

39 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!

40 For Warm Babies and Bears
H+ 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. H+ H+ Heat X H+ H+ H+ Thermogenin (uncoupling protein) H+ H+

41 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.

42 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.

43 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?

44 B. Oxygen will be consumed, but no ATP will be produced.
CQ#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 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.

45 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…. H+ = 10-9 M Intermembrane space [K+] = [Cl-] = 0.1 M H+ = 10-9 M [K+] = [Cl-] = 0.1 M H+ H+ = 10-9 M

46 Can an Artificial Proton Gradient Drive ATP Synthesis?
...into pH 7 buffer, no KCl, with valinomycin present 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. H+ = 10-7 M H+ H+ Intermembrane space H+ H+ Cl- Cl- Cl- H+ Cl- Cl- H+ = 10-9 M K+ K+ [K+] < [Cl-] valinomycin Cl-

47 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 Mitchell’s proposal.

48 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.

49 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."

50 Image Credits Except where noted below, figures appearing in the presentation were created or adapted by author Terry Platt. Slide 24 Description: ATP synthase molecular machine. Source: Image from the Materials Research Science and Engineering Center on Structured Interfaces at the University of Wisconsin-Madison with funding from the National Science Foundation under award numbers DMR , DMR and DMR Any opinions, findings, and conclusions or recommendations expressed in this report are those of the authors and do not necessarily reflect the views of the Foundation.“ Clearance: Used with permission. Slide 28 Description: Schematic diagram of the mitochondrial electron transport chain. Source: Wikimedia Commons, by user Fvasconcellos, Clearance: Released into the public domain by the image author.

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