1. Requirements for oxidative phosphorylation 1. An ion impermeable membrane 2.A mechanism for moving protons (H + ) across the membrane to produce an energy-rich proton gradient 3.A mechanism to capture the energy made available as protons move down the proton gradient Requirements for the production of ATP

2. Requirements for oxidative phosphorylation 1. An ion impermeable membrane See Fig 14.6 in Horton

3. A typical representation of an electron transport chain. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.www.sinauer.comwww.whfreeman.com

4. Requirements for oxidative phosphorylation 2.A mechanism for moving protons (H + ) across the membrane to produce an energy-rich proton gradient Electron transport chain 44 2 4 2H +

5. H+H+ H H AMP NMN nicotinamide

6. H FMNH (semiquinone) H FMNH 2 (hydroquinone) H 1 5 Isoalloxazine -H +, -e - 2H + 2e -

7. H H FADH 2

8. - Semiquinone ( Q - ) H H Ubiquinol ( QH 2 ) Isoprenoid unit + 1 e -, +2H + + 1 e -

9. Non-heme iron sulfur proteins (Fe-S clusters) Cys Fe S SS S S S 2S -2Fe Cys FeS S S S S 4S -4Fe Fe S S S Cys

10. Cytochromes Heme-containing proteins Classified as a, b or c based on absorption spectrum Electron transport has: a and a 3, b 566 (b L ) and b 562 (b H ). and c and c 1 carry 1 electron per heme iron a, a 3, b 566,,, b 562,, and c 1 are integral membrane proteins c is a peripheral membrane protein on outer surface of inner mitochondrial membrane

11. Heme group of Cytochrome b Heme group in cytochromes See fig. 7-34 in Horton methyl proprionate

12. See fig. 7-34 in Horton Heme group in cytochromes a and c

13. See Fig 7-35 -Horton c  nm b  nm a  nm

14. Difference spectra = spectra of experimental– spectra of fully oxidized mito Normal Blocked between Cyt b and c 1 a and c fully oxidised b fully reduced b c a O 2

15. Substrate or complexE o’ (V) NADH-0.32 Complex I FMN Fe-S clusters -0.30 -0.25 - -0.05 Succinate+ 0.03 Complex II FAD Fe-S clusters 0.0 -0.26 – 0.00 QH 2 /Q+0.04 Complex III Fe-S clusters Cyt B 560 Cyt b 566 Cyt c 1 +0.28 - 0.1 + 0.05 + 0.22 Cytochrome c+ 0.23 Complex IV Cyt a Cu A Cyt a 3 Cu B + 0.21 + 0.24 + 0.39 + 0.34 O2O2 + 0.82 Standard redox potentials of mitochondrial oxidation-reduction components

16. Electron transport complexes Complex I: NADH-Q reductase  E 0' = + 0.32 V  G = -70 kJ/2e - Contains at least 34 polypeptides FMN, 2Fe-2S and 4Fe-4S clusters, tightly bound CoQ Mr = 880kD Result: 2 e - from NADH to CoQ 4 H + from matrix to intermembrane space NADH + H + + CoQ ox NADH + CoQH 2 FMN FMNH 2 Fe-S Q QH 2 2e - 2H + 4H + NADH + H + NAD matrix Intermembrane space

17. Complex II: succinate dehydrogenase/ succinate – Q reductase  E 0' = + 0.015 V  G = -2.9 kJ/2e - Contains: FAD, Fe_S Result: 2 e - from FADH 2 to CoQ No protons translocated FADH 2 + CoQ ox FADH + CoQH 2 FAD Fe-S Q QH 2 2e - 2 x e - 2H + succinate Fumarate + 2H + 2 x e - matrix Intermembrane space

18. Complex III: Cytochrome C reductase  E 0' = + 0.25 V  G = --37 kJ/2e - Contains: Cyt b (b L and b H ), Cyt c 1, Fe-S protein, several additional proteins Result: 2 e - from CoQH 2 to Cyt c 2 H + taken up from matrix, 4 H + to intermembrane space CoQH 2 + 2Cyt c (Fe +3 ) CoQ + 2Cyt c (Fe +2 ) 2H + 2 x e - c 4H + QH 2 Q 2H + matrix Intermembrane space

19. C1C1 bLbL Fe-S bHbH C1C1 bLbL bHbH C1C1 bLbL bHbH C1C1 bLbL bHbH CoQ cycle

20. Complex IV: Cytochrome oxidase  E 0' = + 0.57 V  G = -110 kJ/2e - Contains: 10 subunits, Cyt a and Cyt a 3, 2 Cu (A, B) Result: 4 e - from 4 Cyt c to form 2 H 2 O 8 H + taken up from matrix, 4 H + to intermembrane space 4Cyt c (Fe +2 ) + 4 H + + O 2 4Cyt c (Fe +3 ) + 2 H 2 O a - Cu A 1/2O 2 2 x e - 2H + 2 x e - c a 3 - Cu B 2H + H2OH2O matrix Intermembrane space

21. Cu 2+ Fe 3+ Cu + Fe 2+ Cytochrome oxidase: electron transfer to O 2 a3a3 Cu B Cu 2+ Fe 3+ O-O- O-O- Cu + Fe 3+ O-O- O-O- Cu 2+ Fe 3+ O-O- O- H Cu 2+ Fe 3+ OH -.. See Horton, fig 14.16

22. Electron transport chain ComplexMatrixIntramembrane space I-5+4 III-2+4 IV-4+2 II00 Summary of protons translocated per 2 e - Cplx I Cplx IV CoQ Cyt c e-e- 2H + + ½ O 2 H2OH2O NADH + H + NAD Cplx III H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+

24. Paul D. Boyer John E. Walker Jens C. Skou "for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)" "for the first discovery of an ion-transporting enzyme, Na+, K+ -ATPase" 1997 Nobel Prize for Chemistry

25. ATP synthase These and the following images can be found at the home page of Boris A. FenioukBoris A. Feniouk (http://www.biologie.uni-osnabrueck.de/biophysik/Feniouk/Home.html) that contains a wealth ofhttp://www.biologie.uni-osnabrueck.de/biophysik/Feniouk/Home.html additional information on ATP synthesis. See also Horton p 450 and 451.

27. Binding-change mechanism (See page 451 in Horton) From: http://www.cse.ucsc.edu/~hongwang/ATP_synthase.html

28. http://www.res.titech.ac.jp/~seibutu/ For movies and details go to:

29. Cplx I Cplx IV CoQ c c e-e- 2H + + ½ O 2 H2OH2O NADH + H + NAD Cplx III H+H+ H+H+ H+H+ ATP synthase H+H+ ADP + PO 4 ATP H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+