1. Aerobic respiration Mitochondrial structure and function –Visible under light microscope –Universal in aerobic eukaryotes –Have own DNA and ribosomes –Number and shape vary widely in different cell types Number: more in cells with higher E requirements Shape: can undergo fission and fusion to yield typical ‘cylinder’ shape or more complex tubular networks

2. Aerobic respiration Mitochondrial structure and function –Membranes Outer: permeable to many things –Porins, large central pore Inner: highly impermeable –Channels for pyruvate, ATP, etc

3. Aerobic respiration Mitochondrial structure and function –Membranes Outer: permeable to many things –Porins, large central pore Inner: highly impermeable –Channels for pyruvate, ATP, etc Cristae –Complex invaginations of the inner membrane –Functionally distinct –Joined to inner membrane via narrow channels

4. Aerobic respiration Mitochondrial structure and function –Intermembrane space Between inner and outer membranes Also within the cristae Acidified ( high [H + ] ) by action of the Electron Transport Chain (ETC) –H + are pumped from matrix into this compartment –ATP synthase lets them back into the matrix

5. Aerobic respiration Mitochondrial structure and function –Matrix Compartment within the inner membrane Very high protein concentration ~500mg/ml Contains: –ribosomes and DNA –Enzymes of TCA cycle, enzymes for fatty acid degradation

7. NADH enters the mitochondria by one of two mechanisms: 1. aspartate-malate shuttle NADH --> NADH 2. glycerol phosphate shuttle NADH --> FADH 2 Pyruvate to TCA

8. Oxidation-reduction potentials Reducing agents give up electron share –The lower the affinity for electrons, the stronger the reducing agent NADH is strong, H2O is weak Oxidizing agents receive electron share –The higher the affinity for electrons, the stronger the oxidizing agent O2 is strong, NAD + is weak Couples –NAD + - NADH couple (weak oxidizer, strong reducer) –O2 - H2O couple (strong oxidizer, weak reducer)

9. NADH is a stronger reducing agent than FADH2 strong oxidizing strong reducing NADH --> H2O  G 0 ’= -52kcal/mol 7ATP (max), ~3ATP (real) FADH2 --> H2O  G 0 ’= -36kcal/mol 5ATP (max), ~2ATP (real)  G = -nFE

11. 2Pyruvate + 8NAD + + 2FAD + 2GDP + 2Pi --> 6CO2 + 8NADH + 2FADH2 + 2GTP Adding in products of glycolysis, 2NADH + 2ATP Total yield for both: 10NADH + 2FADH2 + 4ATP = 38 ATP How NADH from cytoplasm are counted changes the theoretical yield The Tricarboxylic Acid (TCA) cycle (Kreb’s cycle)

12. In two steps: –A dehydrogenase step 3C + NAD  2C + CO2 +NADH –Yields Acetyl group bonded to CoenzymeA (CoA) –A synthase step –2C + 4C(OA)  6C Formation of a tricarboxylic acid from pyruvate (OA)

13. The Tricarboxylic Acid (TCA) cycle (Kreb’s cycle) 2C+4C(OA)  6C 6C+NAD  5C+CO2 +NADH 5C  4C+CO2 +NADH 4C+GDP  4C+GTP 4C+FAD  4C+FADH2 4C+NAD  4C(OA) +NADH

14. Fatty acid catabolism Enzymes localized to mitochondrial matrix –Fatty acids cross inner membrane and become linked to HS-CoA –Each turn of cycle generates FADH2 + NADH2 + Acetyl-CoA

15. Amino acid catabolism Enzymes in mitochondrial matrix –cross inner membrane via specific transporters –Enter TCA at various points

16. General outline of oxidative phosphorylation

17. Electron Transport Chain: e- carriers Electron carriers –Flavoproteins (FMN) –Ubiquinone (Q or UQ) –Cytochromes (b, c1, c, a) –Cu atoms –Fe-S centers –Proton movement driven by complexes I, III, IV coupled to large  E

18. Electron carriers: Ubiquinone Lipid soluble Dissolved within inner mitochondrial membrane Free radical intermediate Free radical ‘escape’ from electron transport chain can damage proteins, lipids, RNA, and DNA in a cell Q UQ

19. Electron Transport Chain Complex I passes e - from NADH to Q and pumps 4H+ out of matrix Complex II passes e - from FADH2 to Q UQ shuttles e - to Complex III

20. Electron Transport Chain Complex III passes e - to Cytochrome c and pumps 4H+ out of matrix Cytochrome c passes e - to Complex IV Complex IV passes e - to O2 forming H2O and pumps 2H+ out 1 pH unit diff

21. ATP synthesis: The ATP Synthase enzyme F1 head/sphere (ATPase) catalyzes ADP + Pi ATP F0 base embedded in inner membrane (H + pass through this) F0 + F1 = ATP synthase –Connected via two additional proteins Central rod-like gamma subunit Peripheral complex (abd) holds F1 in a fixed position –Location Bacteria = plasma mem Mitochondria = inner mem Chloroplast = thylakoid Intermembrane space matrix H+H+ ATP

23. Binding Change mechanism of ATP Synthase Each F1 active site progresses through three distinct conformations –Open (O)  Loose (L)  Tight (T) –Conformations differ in affinity for substrates and products Central gamma (  ) subunit rotates causing conformation changes

24. Rotational catalysis by ATP synthase 1 pH unit diff Central gamma (  ) subunit rotation caused by proton (H+) translocation drives the conformation changes

25. Rotational catalysis by ATP synthase If true, should be able to run it backwards (ATP --> ADP + Pi) and watch gamma spin like a propeller blade

26. Rotational catalysis by ATP synthase

27. Other fxns of electrochemical gradient E also used for: –Import of ADP + Pi (+H + ) and export of ATP –Import of pyruvate (+H + ) Uncoupling sugar oxidation from ATP synthesis –Uncoupling proteins (UCP1-5) UCP1/thermogenin, shuttles H + back to matrix (endothermy) –Brown adipose tissue »Present in newborns (lost with age) and hibernating animals »Generates heat –2,4-dinitrophenol (DNP) Ionophore that can dissolve in inner membrane and shuttle H + across –1930’s stanford diet pill trials: overdose causes a fatal fever