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 –Rich in cardiolipin (also present in bacterial membanes) –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 –Rich in cardiolipin (also present in bacterial membanes) –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

6. Glycolysis –6C glucose --> --> --> 2x3C pyruvate + 2NADH

10. Glycolysis –6C glucose --> 3C pyruvate + 2NADH 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

11. The Aspartate – Malate Shuttle

12. TCA cycle –3C --> 2C + CO2 + NADH –CoA derived from pantothenic acid –Condensation: 2C + 4C --> 6C –Isomerization

13. TCA cycle –3C --> 2C + CO2 + NADH –CoA derived from pantothenic acid –Condensation: 2C + 4C --> 6C –Isomerization –6C --> 5C + CO2 +NADH –5C --> 4C + CO2 +NADH –4C + GTP –4C + FADH2

14. TCA cycle –3C --> 2C + CO2 + NADH –CoA derived from pantothenic acid –Condensation: 2C + 4C (OA) --> 6C –Isomerization –6C --> 5C + CO2 +NADH –5C --> 4C + CO2 +NADH –4C + GTP –4C + FADH2 –Hydration –4C (OA) + NADH

15. TCA cycle 2Pyruvate + 8NAD + + 2FAD + 2GDP + 2Pi --> 6CO2 + 8NADH + 2FADH2 + 2GTP –Adding in products of glycolysis, 2NADH + 2ATP –Total yield of glycolysis and TCA cycle: 8NADH + 4FADH2 + 4ATP

16. 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

17. Amino acid catabolism Enzymes in matrix –AA’s cross inner membrane via specific transporters –Enter TCA at various points

18. General outline of oxidative phosphorylation

19. 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)

20. Oxidation-reduction potentials Eo’ measures affinity for electrons –Negative Eo’ indicates a stronger reducing agent –Positive Eo’ indicates a stronger oxidizing agent –In Electron Transport Chain: e - are passed from stronger reducing agents to form weaker reducing agents Pass from more negative to more positive Eo’ H2O is the weakest reducing agent (of interest here) NADH --> H2O  G 0 ’ = -53kcal/mol 7ATP (max), ~3ATP (real)

21. Electron Transport Chain Electron carriers –Flavoproteins FAD/FMN (riboflavin) –NADH DH (complex I) –Succinate DH (complex II, TCA cycle)

22. Electron Transport Chain Electron carriers –Flavoproteins FAD/FMN (riboflavin) –NADH DH (complex I) –Succinate DH (complex II, TCA cycle) –Cytochromes Heme (Fe) groups

23. Electron Transport Chain Electron carriers –Flavoproteins FAD/FMN (riboflavin) –NADH DH (complex I) –Succinate DH (complex II, TCA cycle) –Cytochromes Heme (Fe) groups –Cu atoms Cu 2+ Cu 1+ –Ubiquinone Free radical intermediate Lipid soluble Dissolved within inner mitochondrial membrane –Fe-S centers

24. Electron Transport Chain Electron carriers –Flavoproteins FAD/FMN (riboflavin) –NADH DH (complex I) –Succinate DH (complex II, TCA cycle) –Cytochromes (b, c1, c, a) Heme (Fe) groups –Cu atoms Cu 2+ Cu 1+ –Ubiquinone (Q or UQ) Free radical intermediate Lipid soluble Dissolved within inner mitochondrial membrane –Fe-S centers

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

26. 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

27. 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 –The two pieces are connected via two additional proteins Central rod-like gamma subunit Peripheral complex that holds F1 in a fixed position –Location Bacteria = plasma mem Mitochondria = inner mem Chloroplast = thylakoid 1 pH unit diff Intermembrane space matrix H+H+ ATP

29. The ATP Synthase mechanism Binding Change Mechanism –E of H+ movement is used to force release of ATP from enzyme Enz-ADP + Enz-Pi --> Enz-ATP  G 0 ’ ~ 0 –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 1 pH unit diff

30. 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 1 pH unit diff

31. 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 1 pH unit diff