Case 9- DNP & Oxidative Phosphorylation

What is the site of oxidative phosphorylation?

The inner mitochondrial membrane

What is the site of the citric acid cycle?

The mitochondrial matrix

Why does oxidative phosphorylation happen in the inner mitochondrial membrane?

The high surface area from its folds create more sites for oxidative phosphorylation to occur

Define reduction potential (E₀^’) in your own words

Reduction potential is an affinity for electrons much like electronegativity but compared to hydrogen (which is set at 0). If a substance is more electronegative than hydrogen, it has a positive redox potential. If a substance is less electronegative than hydrogen, it has a negative redox potential.  

What enables electrons to move from NADH or FADH₂ to complex I, to the other complexes, to molecular oxygen?

Electrons move from NADH or FADH2 to complex I and other complexes since electrons move from compounds with lower reduction potential to ones with higher reduction potential. Since oxygen has a positive reduction potential and thus a high affinity for electrons, the electrons flow through the complexes to molecular oxygen.

Explain how electron transfer leads to ATP production.

Electrons are transferred along the electron transport chain which generates the force to move protons from the cytoplasm to the inner mitochondrial matrix. These protons are moved into the mitochondrial matrix via ATP synthase. ATP production is then powered by the proton-motive force generated by ATP synthase.  

Metformin is a weak inhibitor of Complex I and is currently prescribed for the treatment of type 2 diabetes. How might this drug exert its anti-diabetic effects? 

The inhibition of complex I lowers the amount of ATP created by the electron transport chain. This creates a need for more NADH and FADH2 for the citric acid cycle to run, resulting in the use of more acetyl CoA which requires the use of pyruvate and glucose. This results in increased glycolysis to fund this pyruvate need, resulting in less glucose in the blood. 

Explain how electrons from cytoplasmic NADH enter mitochondria to use as electron donors in electron transport chain.

They enter through one of two shuttles. The first shuttle is the glycerol 3-phosphate shuttle used  in the muscle which produces FADH2 meaning it generates 1.5 ATP per NADH. The second shuttle is the malate aspartate shuttle used in the heart and liver which produces NADH so it generates 2.5 ATP. Electrons are transferred to dihydroxyacetone phosphate from NADH. The glycerol 3-phosphate shuttle involves cytoplasmic glycerol 3-phosphate dehydrogenase which oxidizes NADH. Glycerol 3-phosphate is reformed so that FAD can be reduced to FADH2, allowing electrons to be transferred to Q and continue along the electron transport chain.  

How many moles of ATPs are formed when one mole of glucose is completely oxidized to CO₂ and H₂O? 

      Show your work in detail.  

30 to 32 ATP could be formed depending on where in the body it is occurring.  

ATP- 2 net gain from glycolysis, 2 from the two citric acid cycles: Initial total of 4 

NADH- 2 from glycolysis, 2 from the PDH complex, 6 from the CAC: Initial total of 10 

- 10 (2.5) = 25 ATP 

FADH2- 2 from the CAC: initial total of 2 

- 2 (1.5) = 3 ATP 

25 + 3 + 4 = 32 ATP  
Or 30 ATP if NADH is oxidized  

What is the predominant form of DNP at neutral pH?

Deprotonated

Would DNP directly affect electron flow? Why or why not?  

DNP would directly affect the electron flow because it has the ability to carry hydrogen ions across the inner mitochondrial membrane. This decreases the amount of hydrogen ions that will ultimately go through the electron transport chain, reducing the proton-motive force, and reducing the amount of ATP able to be created.  

Propose a mechanism for the effect of DNP on oxidative phosphorylation.

DNP dissipates proton motive force in the electron transport chain and thus results in oxidative phosphorylation in being uncoupled. This happens because DNP is lipid soluble and has a pKa of 4.1. This allows it to lower the efficiency of the electron transport chain by decreasing the proton gradient since it carries protons and moves them across the inner membrane of the mitochondria, removing them from the intermembrane space.  

Explain how DNP cause weight loss?

DNP causes weight loss because its presence causes the electron transport chain to create less ATP, lowering energy charge. This low energy charge and lack of ATP stimulate the body into increasing glycolysis to increase the citric acid cycle function and fatty acid metabolism to create the ATP its missing. This increase in glycolysis results in an increased use of glucose followed by a potential increased use of fat storage to get enough energy to continue these processes.  

Why did the patient’s temperature increase?  

The increase in temperature is due to the electron transport chain becoming nonfunctional. Although energy from proton motive force would normally be transformed into kinetic energy in ATP synthase then chemical energy in the form of ATP, it ends up being released as heat instead. 

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.

The electron transport chains activity ends up needing to be increased since DNP lowers the number of protons in the inner membrane space. This increase in need for protons causes an increase in need for oxygen, increasing the respiratory rate. The oxygen concentration increase is a result of oxygen having the highest reduction potential.  

Why would the patient be experiencing widespread muscle rigidity? Consider what is needed in order for muscles to function normally. 

DNP can cause muscle rigidity due to the lack of ATP available for the muscle to use. DNP lowers hydrogen ion concentration, which decreases the amount of ATP available for processes like muscle contraction which involves ATP binding to myosin heads. 

How did DNP cause this patient’s death? 

 

The patient's death was caused by a lack of ATP making it to their heart due to DNP decreasing hydrogen ion concentration and the muscle rigidity cause by it. The increase in body temperature from DNP also contributed to the patient's death as high temperatures cause proteins to denature, allowing cells to die.  

A recent report from Gerry Shulman’s group at Yale is getting a lot of attention to DNP. Shulman is one of the world’s experts on diabetes and metabolism, and his lab has been working on DNP for some years now. 100-fold less than the standard human dose does a dramatic job of reversing diabetes symptoms in rodent models, and fatty liver disease as well. Suggest a biochemical rational for this proposal.

A small dosage of DNP would still decrease the amount of available ATP and cause an increase in need for glycolysis to attempt to make up for this lack of energy. It being such a small dosage would allow this increase in need for ATP to be helpful for patients with type 2 diabetes by using up the extra glucose in their blood to fuel glycolysis rather than being deadly. If enough glucose is used up, it could even increase fat degradation, resulting in the decreased fatty liver disease as well.  

Define “brown fat.” 

Brown fat’s major function is thermoregulation. It is stored in brown adipose tissue with many mitochondria and uncoupling proteins. 

Describe how brown fat helps an organism stay warm.  

Brown fat helps an organism stay warm due to the uncoupling proteins in their mitochondria. Similarly to the function of DNP, these uncoupling proteins transport protons from the intermembrane space into the matrix and generate heat energy rather than allow energy to be captured by ATP. 

Where does fatty acid oxidation take place?

The mitochondrial matrix

Oxidative phosphorylation is a series of what that does what?

Oxidative phosphorylation is a series of oxidation-reduction reactions that allow the flow of electrons from NADH and FADH2 to oxygen

Is the electron flow occurring in the electron-transport chain exergonic or endergonic?

Very exergonic

How many of the 4 large protein complexes involved in the electron transport chain use the energy released by the electron flow to pump protons fro the mitochondrial matrix into the space between the inner and outer mitochondrial membranes?

3

What is the proton gradient created by the electron transport chain used for?

To power the synthesis of ATP

Which of the complexes in the electron transport chain is not actually a transmembrane protein?

Complex II

Describe the structure of ATP synthase

Part of the enzyme is imbedded in the inner mitochondrial membrane while the rest of it resides in the matrix. Each proton enters the cytoplasmic half-channel, follows a complete rotation of the c ring, and exits through the other half channel into the matrix.

Is NAD+ to NADH oxidation or reduction?

Reduction

What is the driving force of the ETC?

Reduction potential

What has the greatest reduction potential?

Oxygen

Does a reductant lose or gain electrons?

Lose electrons

Does an oxidant lose or gain electrons?

Gain electrons

What does a positive reduction potential say about a species affinity for electrons?

The more positive the potential, the greater the species’ affinity for electrons and tendency to be reduced

What kind of oxidants gain electrons very easily?

Strong oxidants like oxygen

Describe a substances redox potential in relation to electronegativity

Substances more strongly electronegative than hydrogen have positive redox potentials and substances less electronegative than hydrogen have negative redox potentials

What happens when electrons flow “downhill” in a redox reaction?

They release free energy

What is the driving force of oxidative phosphorylation?

The electron-transfer potential of NADH or FADH2 relative to that of O2

Why does FADH2 go to complex II?

Complex II is Succinate dehydrogenase seen in the citric acid cycle

What is the only enzyme that participates in both the citric acid cycle and the electron transport chain?

Succinate dehydrogenase (Complex II)

How many moles of ATP are generated by one mole of NADH?

2.5 moles ATP

How many moles of ATP are generated by one mole of FADH2?

1.5 mole ATP

With what shuttle and where in the body will NADH be turned into FADH2 after passing from the cytoplasm to the mitochondria?

The glycerol 3-phosphate shuttle and in the muscle

With what shuttle and where in the body will NADH remain NADH after passing from the cytoplasm to the mitochondria?

The malate-aspartate shuttle in the heart and liver

Is DNP lipid soluble or water soluble?

Lipid soluble

Describe the molecular mechanism of DNP poisoning in comparison to normal physiological conditions.

Under normal conditions, the ETC and Ox. Phos. are coupled by proton motive force which is used to synthesize ATP. When DNP poisoning occurs, it dissipates the proton motive force, causing ETC and Ox. Phos. to be uncoupled. With proton motive force gone, no force pushes H+ through ATP synthase, meaning less ATP is made.

What are the 4 fates for energy released from a biochemical reaction in a living organism?

1. Transfer to another chemical (stored in new bonds)

2. Store as potentials (i.e, proton motive force, ATP)

3. Work (i.e, conformational changes)

4. Heat (support body temperatures)