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,1,The “Energy Burner”,“Cheryl! Come here!” Charles called to his twin sister excitedly.“What?” as
2、ked Cheryl.“You know how my JV wrestling coach wants me to get down to the 145-pound weight category? Well, its here!”“Whats here?”“The solution! DNP!”,2,Just a Few Little Pills,Cheryl picked up the invoice. “Seventy-one bucks for 11 tablets? What is musle- anyhow?”Charles nodded. “Its not cheap, bu
3、t 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?”,3,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, an
4、d 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
5、to win, and this is my first college match!”,4,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
6、 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”,5,You Can Lose More Than Weight,“I feel kind of nauseous, too, and weak in the knees,”
7、 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 imme
8、diately called an ambulance for emergency treatment, as they recognized that there was more than Charles weight at stake.,6,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 serio
9、us 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.,7,A Diet You May Die For,“M
10、edically, 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 highe
11、r 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!”,8,Charles is Alive and Stable,While Charles was recovering, Cheryl found a paper “Dying to be thin: A dinitrophenol related fatality.”* The authors note th
12、at 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 1
13、02 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 patie
14、nt 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.,9,Why Does DNP Do This?,Cheryl read some more. In an article titled “Weight loss and 2,4-dinitrophenol poisoning,”*a 27-
15、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 ca
16、uses 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
17、-567.,10,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?,11,CQ#1: Choose the
18、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 biologic
19、al molecules. Synthesis of ATP is dependent on the passage of electrons through the electron transport chain.,12,Heres a Quick Review,“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!”
20、“.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
21、 combine in the end with molecular oxygen to produce water. But thats only the first half, the oxidative part, of the story.”,13,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
22、 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 inor
23、ganic phosphate (usually termed Pi). Thats the second half.”,14,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
24、 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.,15,Some Questions,“Lets
25、 see if you have the basics, Cheryl,” Dr. Adams proposed. “First, Im sure you know that cyanide is a nasty poison thats 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
26、 Phos?”,16,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
27、 predict?,17,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.,Conclusion: Cyanide prevents oxygen consumption, and because ATP prod
28、uction is also prevented, ATP synthesis must require electron transport.,Cyanide inhibits Complex IV, known as cytochrome oxidase, preventing the transfer of electrons to molecular oxygen.,Time,ATP synthesized,Oxygen consumed,Cyanide added,Succinate added,ADP + Pi added,18,The answer is A.,Another E
29、xperiment,“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”,19,CQ#3: You incubate isolated intact mitochondria in a buffered solution with succinate (an o
30、xidizable 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 pr
31、oduced. Oxygen will not be consumed, but ATP will be produced. Oxygen will be consumed, and ATP will be produced.,20,Conclusion: Not only does ATP synthesis require electron transport, but electron transport requires ATP synthesis.,Blocking ATP synthase also prevents the consumption of oxygen. How c
32、an this be?,Time,ATP synthesized,Oxygen consumed,Oligomycin added,Succinate added,ADP + Pi added,21,The answer is A.,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
33、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.”,22,How Does Coupling Work?,For many years, this phenomenon
34、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!,23,Motors in Cells?,Dr. Adams smiled. “Elegant enzy
35、me 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:,24,What About the Protons?,“Thats pretty neat, but where do the protons come fr
36、om 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 wit
37、h 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.”,25,Three Link
38、ed 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
39、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.,26,It Starts to Make Sense,Dr. Adams conti
40、nued: “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
41、at a picture youve seen before.”,27,Peter Mitchells Chemiosmotic Hypothesis,Protons are pumped out (by the ETC) and flow back in (via ATP synthase) to make ATP.,28,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
42、 the ETC and ATP synthesis?” asked Dr. Adams.Cheryl thought, and came up with an idea.What idea do you have for this question?,29,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 inhi
43、bitor), 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 A
44、TP will be produced. Oxygen will not be consumed, but ATP will be produced. Oxygen will be consumed, and ATP will be produced.,30,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, thoug
45、h no ATP is being made. The mitochondria are said to be uncoupled. How is this?,Time,ATP synthesized,Oxygen consumed,Succinate added,ADP + Pi added,DNP added,Uncoupled,Oligomycin added,31,What Does DNP Actually Do?,“Heres the structure of DNP, with a ring that looks sort of like benzene do you think
46、 it would be more soluble in water, or in a hydrophobic solvent?”,32,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 think
47、ing.” 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. S
48、o heres an application to think about”,33,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
49、 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 unc
50、hanged for 2.,34,“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 t
51、hose 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.”,
