1. Cellular metabolism George Howell III, Ph.D

2. Basics of cellular metabolism Uptake of sugars (e.g. glucose) Glycolysis Citric acid cycle Electron transport chain Oxidative phosphorylation

3. Figure 2-86 Molecular Biology of the Cell (© Garland Science 2008) ATP synthase Metabolic overview (aerobic respiration)

4. HOW CELLS OBTAIN ENERGY FROM FOOD Most Animal Cells Derive Their Energy from Fatty Acids Between Meals Sugars and Fats Are Both Degraded to Acetyl CoA in Mitochondria The Citric Acid Cycle Generates NADH by Oxidizing Acetyl Groups to CO 2 Electron Transport Drives the Synthesis of the Majority of the ATP in Most Cells Chapter 2 © Garland Science 2008

5. Figure 2-80 Molecular Biology of the Cell (© Garland Science 2008) Pathways of production of acetyl CoA from sugars and fats Occurs in mitochondrion in eucaryotic cells Produced from both types of major food molecules Where most of the cell’s oxidation reactions occur Where most of the cell’s ATP is made

6. Figure 2-78 Molecular Biology of the Cell (© Garland Science 2008) Stored fats are mobilized for energy production (animals) Low glucose levels in the blood Hydrolysis of triacylglycerol in fat droplets Free fatty acids and glycerol enter bloodstream Bind to serum albumin (blood protein) Fatty acid transporters in fat oxidizing cells pass fatty acids to cytosol To mitochondria for energy production HSL

7. Outline of glycolysis 2 ATP in…..4 ATP out = net gain of 2 ATP 2 molecules of NADH out Phosphofructokinase = rate limiting step Fructose 6 phosphate to fructose 1,6 - bisphosphate

8. Figure 2-70 Molecular Biology of the Cell (© Garland Science 2008) Glycolysis (cont.)

9. 2 pathways for anaerobic breakdown of pyruvate Figure 2-71a Molecular Biology of the Cell (© Garland Science 2008) Inadequate oxygen In some anaerobic organisms

10. Net production: 3 NADH 1 FADH 2 1 GTP 2 CO 2 Pyruvate dehydrogenase = 1 NADH TCA cycle

11. Figure 14-1 Molecular Biology of the Cell (© Garland Science 2008) Harnessing Energy for Life Protons (H + ) Freely available from water molecules The pump harnesses the energy of electron transfer to pump protons Creates a proton gradient across the membrane. (A)The essential requirements for chemiosmosis are: a membrane—in which are embedded a pump protein an ATP synthase a source of high-energy electrons (e - ) (B) The proton gradient Serves as an energy store Used to drive ATP synthesis by the ATP synthase enzyme.

12. Figure 14-5 Molecular Biology of the Cell (© Garland Science 2008) The relationship between mitochondria and microtubules (A)A light micrograph of chains of elongated mitochondria in a living mammalian cell in culture. The cell was stained with a fluorescent dye (rhodamine 123) that specifically labels mitochondria in living cells. (B) An immunofluorescence micrograph of the same cell stained (after fixation) with fluorescent antibodies that bind to microtubules. Note that the mitochondria tend to be aligned along microtubules. Why be highly associated with microtubules??

13. Figure 14-8 Molecular Biology of the Cell (© Garland Science 2008) The general organization of a mitochondrion In the liver, an estimated 67% of the total mitochondrial protein is located in the matrix, 21% is located in the inner membrane, 6% in the outer membrane, and 6% in the intermembrane space. Each of these four regions contains a special set of proteins that mediate distinct functions.

14. Figure 14-10 Molecular Biology of the Cell (© Garland Science 2008) A summary of energy-generating metabolism in mitochondria Pyruvate and fatty acids enter the mitochondrion (bottom) and are broken down to acetyl CoA. The acetyl CoA is then metabolized by the citric acid cycle, which reduces NAD + to NADH (and FAD to FADH 2 ). In the process of oxidative phosphorylation, high- energy electrons from NADH (and FADH 2 ) are then passed along the electron-transport chain in the inner membrane to oxygen (O 2 ). This electron transport generates a proton gradient across the inner membrane, which is used to drive the production of ATP by ATP synthase. The NADH generated by glycolysis in the cytosol also passes electrons to the respiratory chain. NADH cannot pass across the inner mitochondrial membrane the electron transfer from cytosolic NADH must be accomplished indirectly done by means of one of several “shuttle” systems that transport another reduced compound into the mitochondrion. After being oxidized, this compound is returned to the cytosol, where it is reduced by NADH again.

15. Table 14-1 Molecular Biology of the Cell (© Garland Science 2008)

16. Figure 14-11 Molecular Biology of the Cell (© Garland Science 2008) In this process of oxidative phosphorylation, the inner mitochondrial membrane serves as a device that changes one form of chemical bond energy to another Converts a major part of the energy of NADH (and FADH 2 ) oxidation into phosphate-bond energy in ATP. The major net energy conversion catalyzed by the mitochondrion

17. Figure 14-14 Molecular Biology of the Cell (© Garland Science 2008) The general mechanism of oxidative phosphorylation A high-energy electron is passed along the electron-transport chain Some of the energy released is used to drive the three respiratory enzyme complexes that pump H + out of the matrix. The resulting electrochemical proton gradient across the inner membrane drives H + back through the ATP synthase, a transmembrane protein complex that uses the energy of the H + flow to synthesize ATP from ADP and P i in the matrix.

18. Figure 14-26 Molecular Biology of the Cell (© Garland Science 2008) The path of electrons through the three respiratory enzyme complexes During the transfer of electrons from NADH to oxygen (red lines), ubiquinone and cytochrome c serve as mobile carriers that ferry electrons from one complex to the next. Protons are pumped across the membrane by each of the respiratory enzyme complexes.

19. Redox potential changes along the mitochondrial electron- transport chain The redox potential (E′ 0 ) increases as electrons flow down the respiratory chain to oxygen. Electrons flow through a respiratory enzyme complex by passing in sequence through the multiple electron carriers in each complex. Part of the favorable free-energy change is harnessed by each enzyme complex to pump H + across the inner mitochondrial membrane. NADH is not the only source of electrons for the respiratory chain. The flavin FADH 2 is also generated by fatty acid oxidation and by the citric acid cycle. Its two electrons are passed directly to ubiquinone, bypassing NADH dehydrogenase. Cause less H + pumping than the two electrons transported from NADH.

20. Figure 14-29 Molecular Biology of the Cell (© Garland Science 2008) The standard free-energy change, ΔG°, for the transfer of each of the two electrons donated by an NADH molecule can be obtained from the left-hand ordinate. NADH dehydrogenase and cytochrome b-c 1 complexes each pump two H + per electron Cytochrome oxidase complex pumps one H + per electron ΔG = -n(0.023) ΔE′ 0, where n is the number of electrons transferred across a redox potential change of ΔE′ 0 mV

21. Figure 14-24 Molecular Biology of the Cell (© Garland Science 2008) Quinone electron carriers in the respiratory chain Ubiquinone picks up one H + from the aqueous environment for every electron it accepts Ubiquinone can carry either one or two electrons as part of a hydrogen atom When reduced ubiquinone donates its electrons to the next carrier in the chain, the protons are released. A long hydrophobic tail confines ubiquinone to the membrane and consists of 6–10 five-carbon isoprene units, the number depending on the organism. The corresponding electron carrier in the photosynthetic membranes of chloroplasts is plastoquinone, which is almost identical in structure. For simplicity, both ubiquinone and plastoquinone are referred to in this chapter as quinone (abbreviated as Q).

22. Figure 14-27 Molecular Biology of the Cell (© Garland Science 2008) The reaction of O 2 with electrons in cytochrome oxidase Four protons are pumped out of the matrix for each O 2 molecule that undergoes the reaction 4e - + 4H + + O 2 → 2H 2 O.

23. Figure 14-15 Molecular Biology of the Cell (© Garland Science 2008) ATP synthase Both F 1 and F 0 are formed from multiple subunits. A rotating stalk turns with a rotor formed by a ring of 10 to 14 c subunits in the membrane (red). The stator (green) is formed from transmembrane a subunits, tied to other subunits that create an elongated arm. This arm fixes the stator to a ring of 3α and 3β subunits that forms the head. (B) The three- dimensional structure of the F 1 ATPase, determined by x-ray crystallography. This part of the ATP synthase derives its name from its ability to carry out the reverse of the ATP synthesis reaction— namely, the hydrolysis of ATP to ADP and P i, when detached from the transmembrane portion. head portion, called the F 1 ATPase transmembrane H + carrier, called F 0 The three-dimensional structure of the F 1 ATPase, determined by x-ray crystallography. This part of the ATP synthase derives its name from its ability to carry out the reverse of the ATP synthesis reaction— namely, the hydrolysis of ATP to ADP and P i, when detached from the transmembrane portion.

24. Inhibitors of ETC Cyanide Hydrogen cyanide is most toxic (gas at room temp.) – Most common industrial use is in mining industry to dissolve gold and silver – Used in Nazi Germany in gas chambers during Holocaust May have a bitter almond smell Cyanide ion can come from cyanide salts, apricot, peach, cherry pits (contain toxic amounts of cyanide that must be metabolized by intestinal bacteria to release it), fire smoke (especially burning plastics), vapors from industrial metal plating Main MOA is to bind metalloenzymes (cytochrome oxidase) and inhibit oxidative respiration/phosphorylation – Shifts to anaerobic respiration – Death occurs quickly – Causes metabolic acidosis – Tissues with high oxygen demand (brain and heart) are first damaged Rotenone Broad spectrum pesticide and insectide Interferes with electron donation from complex I to ubiquinone Oral ingestion produces GI irritation Can cause conjunctivitis, dermatitis, pharyngitis, rhinitis Possible link with Parkinson’s upon chronic exposure Treatment is symptomatic

25. Figure 14-13 Molecular Biology of the Cell (© Garland Science 2008) The two components of the electrochemical proton gradient The total proton-motive force across the inner mitochondrial membrane consists of … a large force due to the membrane potential (traditionally designated ΔΨ by experts, but designated ΔV in this text) a smaller force due to the H + concentration gradient (ΔpH). Both forces act to drive H + into the matrix.

26. Figure 14-60 Molecular Biology of the Cell (© Garland Science 2008) The organization of the human mitochondrial genome The human mitochondrial genome contains… 2 rRNA genes 22 tRNA genes 13 protein-coding sequences The DNAs of many other animal mitochondrial genomes have also been completely sequenced. Most encode precisely the same genes as humans Gene order is identical for animals that range from mammals to fish

27. An electron micrograph of an animal mitochondrial DNA molecule caught during the process of DNA replication: The circular DNA genome has replicated only between the two points marked by red arrows. The newly synthesized DNA is colored yellow. Figure 14-54 Molecular Biology of the Cell (© Garland Science 2008)