Metabolic modes of energy generation

1. Metabolic modes of energy generation • Respiration – couple substrate oxidation to the ultimate reduction of an extrinsic chemical such as O2, DMSO, etc. • Fermentation – couple substrate oxidation to reduction of internally generated substrates • Photosynthesis – harvest light energy to facilitate electron transport in energy generating mechanism

2. Chemiosmotic Theory • The transmembrane differences in proton concentration are the reservoir for energy extracted from biological oxidation reactions - Peter Mitchell Fermentation utilizes substrate level phosphorylation, more on that later

3. Oxidative phosphorylation and photophosphorylation have similarities and differences • In eukaryotes, both are organellar processes; mitochondria and chloroplast • Both involve flow of electrons through membrane components • Free energy from electron flow is used to pump protons across a membrane • Flow of protons back drives ATP synthesis • In aerobic systems, Oxidative phospho. Reduces O2 to H2O, while photophospo. Can oxidize H2O to O2

4. Oxidative energy generation leads to ATP, water, and oxidized electron carriers

5. Eukaryotic cells

6. Oxidative phosphorylation is a mitochondrial process • Although the mitochondria imports some biomolecules from the cytoplasm, it contains Citric acid cycle enzymes, it’s own genome, etc. Notice the cristae which increase membrane surface area.

7. Electron carriers initiate oxidative phosphorylation • Pools of electrons linked to carriers such as NAD, NADP, FMN, and FAD are generated by catabolic mechanisms (mostly NADH is generated) • Note when depicted as NAD+, the intent is to reflect oxidation state, NOT charge on the molecule

8. Electrons are passed to membrane components • For instance, NADH is oxidized by a membrane bound enzyme NADH dehydrogenase, which subsequently passes electrons to quinones, and so on. • Various steps are linked to proton translocation out of the mitochondrial inner membrane

9. A membrane is a prerequisite for biological energy generation This serves as an impermeable barrier to many solutes such as H+, even H2O. Polar molecules cannot traverse the hydrophobic layer.

10. Cells can alter the fatty acid content of their membranes • Sterols modify fluidity also

11. Ubiquinone (CoQ) is a lipid soluble two electron, two proton carrier • Plants – Plastoquinone • Bacteria – menaquinone Freely diffusible in Lipid bilayer

12. Cytochromes classified on basis of porphyrin ring

13. Another example of a spectroscopic bioassay

14. Iron-sulfur proteins carry electrons and do more… • At least eight iron-sulfur proteins act in mitochondrial electron transfer • Also Fe-S centers have been shown to be sensors of aerobic/anaerobic gene expression

15. Determining the order of electron transfer • Standard reduction potentials • Spectroscopic measure of carrier oxidation • Inhibitors • Rotenone inhibits NADH dehydrogenase • Antimycin A inhibits cytochrome b • Cyanide, azide, or Carbon monoxide inhibit cytochrome oxidase aa3

16. Experimental evidence for electron transfer order

17. The order of electron transfer under aerobic conditions

18. Cytosolic-derived NADH must be shuttled into the mitochondria • Although citric acid cycle and fatty acid oxidation occur in the “right” place (mitochondrial matrix), glycolysis is cytoplasmic and NADH from this pathway must be shuttled into the matrix of the mitochondria (membrane is impermeable to this compound; no transporter) • Glycerol-3-phosphate shuttle • Malate-Aspartate shuttle

19. Glycerol-3-phosphate shuttle • 2 e- from NADH to FADH2 Get only 2 ATP from FADH2 vs 3 from NADH More on that later

20. Malate-Aspartate Shuttle

21. General class of transporters

22. Electron transport is accomplished by multienzyme complexes

23. NADH:ubiquinone oxidoreductase utilizes NADH generated from catabolic reactions • This is a huge protein complex ~900,000 kDa • Electron transfer from NADH to ubiquinone is coupled to the translocation of protons through the protein, with a stoichiometry of 2H+/e- • NADH + H+ + Q  NAD+ + QH2

24. An electron’s path through this complex • Oxidation of NADH transfers two electrons to FMN bound to the enzyme, and releases a proton into the matrix • The electrons are passed from FMN through a series of Fe-S centers the last one being called N-2 (Six in the case of the mitochondrial enzyme, but only four appear to be universally conserved) • N-2 reduces ubiquinone

25. Proton pumping by this complex • Experiments suggest one proton is pumped into the intermembrane space during NADH to N-2 transfer of one electron, and a second proton during N-2 to ubiquinone transfer of one electron • Recall the stoichiometry, 2 protons per electron – this means four protons in total are pumped for each NADH oxidation