1. INTER 111: Graduate Biochemistry

2.  Define electron transport chain, oxidative phosphorylation, and coupling  Know the locations of the participants of the system/pathways  Predict the flow of electrons under standard state conditions when given a redox half equation and know how to calculate the standard state free energy change given the proper equation and half reactions. Be able to predict the spontaneity of a reaction given the reduction potential.  List components of the respiratory chain and the electron carrying molecules. Know the differences between the hemes.  Outline the pathway of electron transport in mitochondria in terms of the transfer of electrons from the reducing equivalents to oxygen.

3.  Describe the mechanism of action of an uncoupler or inhibitor on the electron transfer chain or oxidative phosphorylation.  Recognize the site of inhibition of rotenone, carbon monoxide, antimycin A, and oligomycin and be able to describe the effect of these inhibitors.  Describe and understand the mechanism of how the F o F 1 ATPase complex forms ATP.  Estimate the net potential yield of ATP for each of the entry points into the electron transport system and know why there are discrepancies.

5. Glucose Acetyl CoA R5P Pyruvate NADH + H + and ATP Glycogen disaccharides NADH + H + and FADH 2 and CO 2 aerobic conditions citric acid cycle Electron transport Oxidative phosphorylation O2O2 H2OH2O AT P ADP + P i Acetyl CoA

6. Oxidative phosphorylation  is aerobic (i.e., in O 2 )  is a stepwise process  transfers electrons from reduced carriers to O 2  generates 3 moles ATP for every mole NADH Electron transfer chain  is a series of coupled oxidation-reduction reactions  is catalyzed by membrane-bound proteins on the inner membrane of mitochondria

7. An oxidation-reduction (redox) reaction involves an electron donor and an electron acceptor. The redox potential expresses the tendency of an electron donor to reduce its conjugate acceptor. Under standard conditions (25 o C, pH 7, [donor]=[acceptor]=1 M), the redox potential is E o ’ E o ’ is measured relative to the standard hydrogen electrode. Fe 2+ + Cu 2+ Fe 3+ + Cu + e - donor e - acceptor oxidized donor reduced acceptor

8. Compounds with a large negative E o are strong reducing agents. Compounds with a large positive E o are strong oxidizing agents. Redox pairE o ’ (V) NAD + / NADH FMN / FMNH 2 Cytochrome b Fe 3+ /Fe 2+ 1/2 O 2 / H 2 O -0.32 -0.22 +0.07 +0.82

10. - 0.32 - 0.30 + 0.04 + 0.07 + 0.23 + 0.29 + 0.55 + 0.82 + 0.25 Eo’Eo’ NAD + / NADH FMN / FMNH 2 Fe 3+ S / Fe 2+ S H-Fe 3+ / H-Fe 2+ CoQ / CoQH 2 O 2 / H 2 O H-Fe 3+ / H-Fe 2+ Redox couples Q (mobile) (mobile) Cyto oxidase (Complex IV) Cyto bc 1 (Complex III) NADH-Q reductase (Complex I) NAD + FMN Fe-S centers Coenzyme Q Cyto b (Fe 3+ ) Fe-S centers Cyto c (Fe 3+ ) Cyto a (Fe 3+ ) Cyto a 3 (Fe 3+ ) O2O2 Cyto c (Fe 3+ ) 2 e - transfer

11. cristae matrix inner membrane outer membrane a a a3a3 a c b CoQ FMN NAD + Electron transport chain ATP synthase

12. The electron transport chain conducts a series of oxidation/reduction reactions. The components of the respiratory chain are flavoproteins, ubiquinone molecules, and cytochromes

13.  Formation of NADH  NAD + is reduced to NADH by dehydrogenases in the TCA cycle. Substrate (reduced) Product (oxidized) NAD + NADH + H +  NADH dehydrogenase  Coenzyme Q  Cytochromes

14. bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c Q 2Fe-2S QH 2 FMN 4Fe-4S NADH QH 2 matrix intermembrane space Cu A a a3a3 Cu B 1/2 O 2 + 2 H + H2OH2O F 1 F O synthase Complex IComplex IIIComplex IV NADH dehydrogenase cytochrome bc 1 cytochrome c oxidase

15. NADH + H + NAD + FMN FMNH 2 Fe 2+ S Fe 3+ S CoQ CoQH 2 reduced oxidized reduced oxidized Q 2Fe-2S QH 2 FMN 4Fe-4S NADH QH 2 matrix intermembrane space

16.  Formation of NADH  NADH dehydrogenase  Coenzyme Q  A quinone derivative with long isoprenoid tail Mobile carrier that accepts hydrogens from FMNH2 (complex I) and FADH2 (Complex II). Transfers electrons to complex III  Cytochromes

17. Reduced form of coenzyme Q (QH 2, ubiquinol) Semiquinone intermediate (QH) Oxidized form of coenzyme Q (Q, ubiquinone)

18. matrix intermembrane space bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c QH 2 Heme

19. Cyto c matrix intermembrane space Cu A a a3a3 Cu B 1/2 O 2 + 2 H + H2OH2O Cyt 2+ a 3 Cyt 3+ a 3 Cyt 3+ a Cyt 2+ a Cyt 2+ c Cyt 3+ c H2OH2O 1/2 O 2

20. Three elements for energy transduction: A cellular membrane Exergonic electron transport generates a proton gradient across a membrane Proton gradient furnishes energy for ATP production by ATP synthase Peter D. Mitchell

21. H+H+ H H+H+ H+H+ H+H+ bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c Q 2Fe-2S QH 2 FMN 4Fe-4S NADH QH 2 matrix intermembrane space Cu A a a3a3 Cu B 1/2 O 2 + 2 H + H2OH2O ATP ADP + P i Complex V ATP synthase lower pH and greater positive charge

22. H+H+ H H+H+ H+H+ H+H+ bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c Q 2Fe-2S QH 2 FMN 4Fe-4S NADH QH 2 matrix intermembrane space Cu A a a3a3 Cu B 1/2 O 2 + 2 H + H2OH2O ATP ADP + P i For 1 mol NADH oxidized, 3 mol ATP produced For 1 mol FADH 2 oxidized, 2 mol ATP produced Complex V ATP synthase

23. 3H+3H+ ATP ADP + P i ATP ADP + P i When electrochemical H + gradient is favorable, F 1 F O ATPase complex catalyzes ATP synthesis. If no membrane potential or pH gradient exists to drive the forward reaction, K eq favors the reverse reaction (ATP hydrolysis). 3H+3H+ F1F1 F0F0

24. F 1 subunit present with stoichiometry      and   and  subunits (513 and 460 residues in E. coli) are homologous to one another 3 nucleotide-binding catalytic sites at  /  interface, but involving  residues Each subunit contains ATP, but is inactive in catalysis Mg 2+ binds with adenine nucleotides in both  and  subunits F 0 subunit present with stoichiometry a, b 2, and c 10 F1F1 F0F0       

25. Rotation of the  shaft relative to the ring of  and  subunits directly observed, by attaching fluorescent-labeled actin filament to the  subunit. Noji et al. 1997 Nature 386, 299 The rotation rate is 100 Hz (revolutions/s) ATP-induced rotation occur in discrete 120 o steps. F 1 F O synthase http://www.res.titech.ac.jp/~seibutu/main_.html

26. HEAT bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c Q 2Fe-2S QH 2 FMN 4Fe-4S QH 2 matrix intermembrane space Cu A a a3a3 Cu B Complex V ATP synthase 2,4-DNP