1. Review the structures of NAD and FAD (oxidized and reduced forms) and ATP.
2. Outline the mitochondrial electron transport system showing the major electron carriers.
Make a note of their localization.
3. Describe the path of electrons into and along the electron transport chain. Understand
what drives the directionality of this flow.
Reduction potential (E0) drives the flow of electrons. Each acceptor has a less negative or more positive E0 than the one before it--meaning it is more accepting of electrons, thus driving the flow.
4. Illustrate why FADH2 produces less ATP than NADH.
FADH2 “deposits” its electrons at complex 2 of the ETC. Unlike complex 1, which NADH uses, complex 2 does not pump electrons--thus the ATP that can be generated from FADH2 is less.
5. Explain the chemiosmotic theory.
Chemiosmotic theory says that most of the ATP generated in respiring cells comes from the electricrochemical gradient across the IMM generated by donation of electrons to the ETC by NADH and FADH2.
6. Define membrane potential and explain its role in ATP synthesis and thermogenesis.
7. Explain the role of uncoupling proteins with particular attention to their role in
thermogenesis (brown fat).
Thermogenin (UCP1) allows flow of protons from the intermembrane space to the matrix, thus uncoupling oxidative phosphorylation from the ETC. This results in increased heat production--to maintain body temperature when needed. Noradrenaline, produced in response to cold, stimulates fatty acid oxidation--thus providing more fuel for this uncoupling method of thermogenesis.
8. Describe the mechanism of ATP synthase.
ATP synthase has an F0 intermembrane subunit that allows proton flow across the IMM and uses the energy of this to drive its F1 subunit, which has three subunits that cycle between tight, loose, and open to generate ATP from ADP and Pi.
9. Describe the effects of inhibitors such as rotenone, antimycin, CO, cyanide, 2,4 dinitrophenol, amytal, atractyloside, aspirin and oligomycin on oxygen uptake by the mitochondria. Mark on the outline in #2 where they inhibit the electron transport chain
Rotenone and Demerol inhibit the NADH dehydrogenase in C1
Amytal prevents reduction of CoQ (between C1 and C3)
Cyanide and CO bind the Fe3+ in cytochrome oxidase (a+a3) and prevent its reduction
2,4-dinitrophenol is a lipid soluble proton carrier; it uncouples the ETC and Ox Phos by binding protons and carrying them across the IMM into the matrix; asprin is also an uncoupler
Atractyloside acts on an adenine carrier that exchanges ATP inside the matrix for ADP
Oligomycin, an antibiotic, binds the F0 subunit of ATP synthase and prevents proton flow
10. Explain how the following deficiencies could severely impair ATP synthesis: riboflavin,
Riboflavin is a precursor of FMN/FMNH2, electron carriers used by complex 1 of the ETC
Copper is a component of cytochrome oxidase that helps shuttle electrons to O2 to make water
Iron is an electron carrier for ETC complexes 3 and 4
11. Understand that premature infants maybe at risk for a copper deficiency and summarize
Infants depend on brown fat (uses uncoupled ETC and Ox Phos) for thermogenesis. Without copper, cytochrome oxidase (a+a3) can’t transfer electrons to O2, thus inhibiting the ETC; the infant may suffer anemia and hypothermia
12. Explain the functions of the glycerol-phosphate and malate shuttles. Be able to calculate
the net yield of ATP from the complete oxidation of glucose, taking into account the different shuttle requirements.
Glycerol phosphate shuttle uses glycerol-3-phosphate to oxidize NADH in the cytosol/intermembrane space to NAD+ and reduces a FAD carrier. This regenerates NAD+ for further glycolysis/TCA and brings FADH2 to the site of the ETC. It allows 2 ATP per NADH.
The malate-aspartate shuttle fulfills a similar function to the GP shuttle, but brings NADH to the ETC, allowing an eqivalent energy transfer of 3 ATP per NADH.