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BCMB3100 with Paula Lemons and Takahiro Ito...

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Name (on eLC): 810#:

Dinitrophenol Case

BCMB 3100 SP2018

PLEASE NOTE: You should work with your peers on this assignment, but your answers should be put into your own words, not a copy of others’. Copying the work of other students for your cases, regardless of past or present 3100 courses, is a violation of UGA academic honesty policy and will result in penalties. We have learned how free energy from the hydrolysis of ATP can be used to drive biochemical reactions, which can be coupled to drive flux through biochemical pathways. In this case, we will explore how electron transfer potential, or the potential for a compound to be reduced, is converted to phosphoryl transfer potential. In other words, we will discuss how energy derived from oxidation-reduction reactions is stored as ATP. Useful resources http://www.wiley.com/legacy/college/boyer/0470003790/animations/electron_transport/electron_transport.htm http://pdb101.rcsb.org/motm/72

1. Create a drawing of the electron transport chain. Start with a simplified mitochondrion represented by two ovals. Include and label the following components in your drawing.  Inner and outer mitochondrial membranes  Enzyme complexes, including ATP synthase  Electron flow between different electron carriers, from NADH and FADH 2 to molecular oxygen  What happens to the oxygen when it accepts electrons  Proton Flow

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2. Define the terms below in your own words. (a) oxidizing agent: An agent that readily accepts electrons, leading to a positive reduction potential. (b) reducing agent: An agent that readily donates electrons, leading to a negative reduction potential. (c) reduction potential (E0’): Similar to phosphoryl transfer potential, or ∆G°’, the reduction potential, or E0’, is the electron-transfer potential. Based upon the oxidizing and reducing agents, the reduction potential is either positive or negative and is measured in volts. A positive reduction potential, for example, indicates that a molecule has a high affinity for electrons.

Name (on eLC): 810#: 3. Consider the flow of electrons through the electron transport chain. What makes the flow of electrons in the electron transport chain a thermodynamically favorable process? Why is oxygen the final acceptor of electrons? Hint: The correct answer is not that it’s favorable because the change in free energy is largely negative; that is the definition of favorable. The electron transport chain utilizes a series of oxidation-reduction reactions to drive electron flow. When NADH is oxidized in the first step, the released electrons can continue being passed down the path, reaching the next reduced agent, FMN. FMN is the oxidized by the next carrier, and the released electrons are passed to the next carrier. The process repeats itself until the electrons finally reach and reduce O 2. The electrons flow easily down this path as each subsequent carrier has a more positive reduction potential than the one before it, meaning that each successive component of the chain has a higher electron affinity for the electrons than the one before it. The process is thermodynamically favorable because when O 2 is finally reduced at the end, a substantial amount of free energy can be released (-231.8 kJ). O 2 is the final acceptor of electrons specifically because of this large, positive reduction potential, or high affinity for electrons. Case: A 27-yr-old woman was admitted to emergency services complaining of fatigue, nausea, and excessive sweating. She reported starting a new diet tablet, containing 2,4-dinitrophenol (DNP), which she bought over the Internet a week before her admission. She had doubled the recommended dose for faster results. Past medical history was negative. She was a non-smoker, non-drinker, and had no known allergies. Initial examination revealed an agitated overweight female (BMI 33) with a Glasgow Coma Scale (GCS) of 15 (i.e., fully conscious, or no apparent brain injury). Her airway was clear, respiratory rate (RR) was elevated (60 breaths/minute), oxygen saturation 100% (“normal”), blood pressure (BP) 122/86, and heart rate (HR) 140 beats/minute. Her temperature was 39°C. There were no other significant clinical findings. Six hours after admission, her situation worsened: GCS 14 (eyes opening to command), RR 44, BP 146/110, HR 150 beats/minutes, and temperature 40°C. An hour later, she became asystolic (i.e., no heartbeat). Cardiopulmonary resuscitation was started. It was not possible to ventilate her due to widespread sustained muscle rigidity. After 14 cycles with epinephrine and atropine, she remained asystolic and was declared dead. 4. DNP is lipid soluble and has a pKa of 4.1 (the structure is shown at right). Keeping this information in mind: a) Would DNP directly affect electron flow through ETC complexes? State Yes or No, and explain why. No. DNP only uncouples oxidative phosphorylation from ATP synthesis; electron transport from NADH to O2 would continue as normal. This just means even though electron transport proceeds as normal, the energy released from the protons is not captured in the form of ATP. b) Would DNP directly affect proton movement? State Yes or No, and explain. Yes. DNP is a lipid soluble substance, meaning it is soluble in the inner membrane where electron transport is occurring. DNP works in this area by carrying protons across the inner membrane down their concentration gradient back into the mitochondrial matrix. Because the protons being pumped out by the ETC are continuously being dissipated by DNP, the proton-motive force is not generated. c) Would DNP directly affect ATPase function? State Yes or No, and explain. No, it does not directly affect it. It indirectly affects it because as DNP is transports protons back into the matrix, no proton-motive force is generated. Without a proton-motive force, ATP synthase will not change its conformation, and ATP will not be formed. d) Propose a mechanism for the effect of DNP on oxidative phosphorylation. Electron transport occurs as normal and protons are pumped from the mitochondrial matrix into the inner membrane. However, because DNP is lipid soluble, it easily crosses the inner membrane and grabs the protons that have just been pumped out, bringing them back from the inner membrane into the matrix. This dissipates the proton gradient as there is no difference in pH from the inner membrane and matrix.

Name (on eLC): 810#: Therefore, the proton motive force is continuously dissipated. Because of this, there is no free energy released as the few protons flow through ATPsynthase. Without the energy being released from the proton motive force, there is no energy to drive the phosphorylation of ADP to produce ATP. Mechanism: 1) Electron transport occurs as normal, and protons are pumped from mitochondrial matrix to the inner membrane 2) DNP, a lipid-soluble molecule, continuously crosses the membrane, bringing back protons from the inner membrane back into the mitochondrial matrix. This dissipates the proton gradient. 3) The proton-motive force is dissipated and uncoupled from ATP production. The enery released from protons coming back into the matrix is used to generate heat, not ATP. 5. Why did the patient’s temperature increase? When oxidative phosphorylation is uncoupled from ATP synthesis, due to DNP, heat is generated instead. When the energy from the proton gradient is released, it is no longer captured as ATP. Instead, the protons flow through the uncoupling protein “UCP-1” to the mitochondrial matrix, releasing heat as they flow down the gradient. Because of this, the patient’s temperature increased. 6. Why was the patient’s respiratory rate abnormally high, even though her oxygen saturation was 100%? Consider why oxygen consumption would continue in the presence of DNP. Oxygen consumption is normal as the electron transport chain is unaffected by the DNP; oxygen is still being reduced to H2O at the end like normal. However, the DNP the patient ingested did uncouple the electron transport chain from oxidative phosphorylation, so the energy from the proton-motive force wasn’t used to produce the patient’s normal supply of ATP. To make up for this, the patient’s body began to metabolize via glycolysis and the citric acid cycle, producing ATP in this way. Because the body is going through the citric acid cycle quicker, this is generating a lot of CO 2. To prevent this from building up in the body, the patient is exhaling much faster than normal, causing her respiratory rate to be abnormally high. 7. Why would the patient be experiencing widespread muscle rigidity? Consider what is needed in order for muscles to function normally. Muscles require ATP to function normally. Because DNP causes the uncoupling, ATP is not produced at the end of the electron transport chain. Without ATP, muscles will not be able to function properly, causing them to become very rigid. 8. FDA banned DNP in 1930s given the narrow window between DNP’s ED 50 and LD50. However, nowadays DNP is still packaged under different names of weight loss tablets sold on the black market. Explain why DNP is so effective in losing weight. Because DNP uncouples ATP production from the electron transport chain, ATP is not produced. To make up for the lack of ATP being produced, the body has to produce energy another way. By breaking down stored carbohydrates, proteins, and fats, the body is able to produce ATP through this fermentation process. Because these energy sources are being depleted for ATP production, weight will drop off. This is why DNP is very effective at weight loss, but can also be very dangerous as your body is forced to break down most of its stored energy sources. 9. There are some instances where excess heat generation can be beneficial. Hibernating animals, for instance, need to ensure they stay warm enough throughout the winter, even when they aren’t eating or moving around. Brown adipose tissue helps with this process. [p.396-39, 3 rd ed.] a. What is the difference in biochemical makeup between brown adipose tissue and white adipose tissue? Brown adipose tissue is rich in mitochondria (“brown-fat mitochondria”). These mitochondria contain large concentrations of the uncoupling protein UCP-1, or thermogenin, which is used for heat

Name (on eLC): 810#: generation. In contrast, white adipose tissue doesn’t contain large amounts of mitochondria or UCP-1; it is used only as an energy source, not as a source of thermogenesis. b. What is the physiological function of brown adipose tissue? Provide a biochemical explanation of how it is achieved. Because brown adipose tissue contains large concentrations of mitochondria and UCP-1, it can generate heat in the process of nonshivering thermogenesis, which is why it is considered a specialized tissue. Once the proton-motive force builds up, it flows through UCP-1, a membrane protein, rather than flowing through ATP synthase and generating ATP. Instead, as the protons flow down their gradient through UCP-1 into the matrix, the energy released from the proton-motive force is utilized in heat production. Therefore, brown adipose tissue functions as a way to generate heat. c. Explain how the physiologic function of brown fat tissue or the adverse action of DNP in normal mitochondria are examples of the First Law of Thermodynamics. The First Law of Thermodynamics states that energy is conserved in that it is simply transformed from one form to the next. In brown fat tissue or in the effect of DNP, the energy released from the proton-motive force must be transformed to something else. Because oxidative phosphorylation has been uncoupled from ATP synthesis, the energy released from protons is not captured in the form of ATP. Instead, to continue following the law of thermodynamics, the energy released when protons flow down into the matrix is captured into the form of heat energy....