Aerobic respiration

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 --> FADH2 • 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 DG = -nFE strong reducing NADH --> H2O G0’= -52kcal/mol 7ATP(max), ~3ATP(real) FADH2 --> H2O G0’= -36kcal/mol 5ATP(max), ~2ATP(real) strong oxidizing

11. The Tricarboxylic Acid (TCA) cycle (Kreb’s cycle) 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

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

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 UQ Q

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 ATP matrix Intermembrane space H+

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 • Central gamma () subunit rotation caused by proton (H+) translocation drives the conformation changes 1 pH unit diff

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