1. The rate of electron transfer between groups – the distance between donor and acceptor of electron – the free energy change The typical electron transfer rate if groups in contact: 10 13 /s The distance between electron-carrier groups: 15 A° 15 A°/1.7A° ≒ 9, decrease 10 9  10 4 /s If no protein mediator: 15 A°/0.8A° ≒ 19, decrease 10 19  10 -6 /s ≒ 1 day 0.8A ° 1.7A ° e -  cytochrome c  Ⅲ  Ⅳ

2. inverted region The rate of electron transfer between groups – the distance between donor and acceptor of electron – the free energy change Ch. 19 ?

3. Chemiosmotic hypothesis (1961, Mitchell P.)  NADH oxidation  ADP phosphorylation  a covalent high-energy intermediate or an activated protein conformation  proton-motive force drives the synthesis of ATP by mitochondrial ATP synthase (F 1 F 0 ATPase, complex Ⅴ ) Proton-motive force ﹙  p ﹚ : the pH gradient ﹙  pH ﹚ + the charge gradient [membrane potential ﹙  ﹚ ]

4. Testing the chemiosmotic hypothesis Artificial membrane Respiratory chain ATP synthase Proton gradient (A purple-membrane protein, pump protons when light) (from beef heart) A separate system:

5. 19.2 ATP synthesis Mitchell: chemiosmotic model, proton-motive-force ADP + P i + n H p + → ATP + H 2 O + n H N + a proton pore associated with ATP synthase Nelson

6. ATP synthase mechanism: Mg 2+ require Orthophosphate HPO 4 2-

7. The role of proton gradient is to release ATP from ATP synthase but not to form ATP ADP + Pi + ATP synthase in H 2 18 O isotopic-exchange experiments: enzyme-bound ATP forms readily in the absence of a proton-motive force + ATP synthase

8. ATP synthase structure Two functional components: 1. Moving units c-ring,  stalk 2. Stationary unit matrix (  3,  3, , and  )  subunits participate directly in ATP synthesis proton channel complex c-ring (10~14 c subunits) 1a, 2b, 1 

9. Boyer PD (2000): binding-change mechanism  subunit make the 3  subunits unequivalent Loss The conformation of binding ADP and Pi Tight The conformation of binding ATP Open The conformation of releasing ATP

10. A counterclockwise direction in  subunit Binding-change mechanism: T L O Proton drive 

11. The smallest molecular motor Fluorescence labeled Polyhistidine tags of N-terminal of  subunit Nicklel ions are coated on glass surface Only cloned  3  3  subunits  The  subunit was rotated, driven by the hydrolysis of ATP  120 ° increment/ATP hydrolysis  Near 100% efficiency

12. Components of the proton-conducting unit of ATP synthase  Two hydrophilic half-channel  Do not span the membrane  Directly about one c subunit, separately Asp 61 a pair of  -helices COO - /COOH The site of proton entrance

13. Proton flow/c-ring rotation power  rotation, then ATP synthesis Arg 210 in subunit a (02), Ex. 18 [H + ] cyto /[H + ] matrix  25 hydrophobic interaction

14. Proton path through the membrane H+H+  C ring tightly links to  and  subunits C ring rotate   rotate  360 °/3 ATP 10 c subunits/3 ATP  3.33 protons/ATP NADH: 10 H +, 2.5 ATP FADH 2 : 6 H +, 1.5 ATP Ex. 19

15. 116 watts (joule/s) provides energy to sustain a resting person 921 earthquake

16. § 18.5 Shuttles: an array of membrane-spanning transporter proteins Glycerol 3-phosphate shuttle: electrons of cytosolic NADH from glycolysis enter mitochondrial electron transport chain especially prominent in muscle and some insects lack lactate dehydrogenase G3P  1,3bisP  against NADH gradient  1.5 ATP formation for 1 NADH from glycolysis

17. Malate-aspartate shuttle  in heart and liver  is readily reversible  the NADH/NAD + ratio of the cytosol is higher than that of mitochondria  2.5 ATP formation for 1 NADH from glycolysis transamination

18. ATP-ADP translocase (adenine nucleotide translocase or ANT)  highly abundant in the inner mitochondrial membrane (15%)  30 kd, a single nucleotide-binding site, without Mg 2+ ﹙ Ex. 20 ﹚  ADP first entry then coupled to ATP exit, even though the transport rate of ATP is 30-fold higher than that of ADP  high energy consumed, about ¼ of the energy from e - transfer Bongkrekic acid Membrane potential Proton-motive force Ex. 22 P site N site atractyloside

19. ATP synthasome: ATP synthase, ATP-ADP translocase, phosphate carrier (electroneutral exchange) H 2 PO 4 - Mitochondrial transporters Dicarboxylate carrier  40 genes in human genome are encoded

20. Consumed a proton during ATP translocation Other metabolites translocated  2e - transfer less than 10H + formation or +5 Glucose is completely oxidized Glycolysis + 2 TCA cycle (GTP) + 2 or 32 anaerobic metabolism: 2 ATP

21. the rate of oxidative phosphorylation is determined by the need for ATP Respiratory control (or acceptor control) the rate of oxidative phosphorylation is primarily regulated by ADP level

22. Energy charge regulation The rate of TCA cycle is controlled by the availability of NAD + and FAD ATP synthasome

23. Uncoupling proteins (UCPs)  dissipate proton flow  UCP1 (thermogenin) Temp.    -adrenergic agonists  triacylglycerol degrade  free fatty acids liberate  Activate UCP-1  generate heat

24. UCP proteins:  generate heat to maintain body temperature in hibernating animals, some newborn animals, and in mammals adapted to cold  Brown adipose tissue (also brown fat mitochondria), which is very rich in mitochondria, is specialized for nonshivering thermogenesis (vs. white adipose tissue)  regulate the body weight (obesity)[UCP2 and UCP3]  increase the evaporation of odoriferous molecules, skunk cabbage p. 533 7 th line: greenish-colored cytochromes

25. Uncoupler  disrupted the coupling of electron transport and phosphorylation – dissipated proton-motive force  oxygen consumption, NADH oxidation, no ATP formation  heat loss  DNP and certain other acidic aromatic compounds used in some herbicides, fungicides, weight- loss drug (?)

26. Sites of action of inhibitors of electron transport ferric ferrous form of heme a 3

27. Oligomycin Dicyclohexylcarbodiimide (DCCD) prevent the influx of protons through ATP synthase

28. Alternative mechanisms in plant mitochondria Araceae: one family of stinking plants thermogenesis a cyanide-resistant QH 2 oxidase bypass complex III and Ⅳ a rotenone insensitive NADH dehydrogenase, bypass complex Ⅰ a skunk cabbage Nelson

29. Mitochondria ¤ semiautonomous organelles ¤ endosymbiotic double membrane, circular DNA, specific transcription and translation machinery ¤ Maternally inherited

30. Mitochondria – diseases  a center of energy metabolism  Leber hereditary optic neuropathy – NADH-Q oxidoreductase (complex Ⅰ ) mutation – resulted in blindness during midlife  chance fluctuation percentage  the threshold of defect  the accumulation of mutations  effect the energy transduction, reactive oxygen species (ROS) generation  nervous system and heart are vulnerable – aging, degenerative disorders, and cancer.

31. Three mitochondrial cell death pathways Apoptosis inducing factor (AIF) Apoptotic protease activating factor-1(Apaf-1) mtPTP (mitochondrial permeability transition pore) Programmed cell death PS Cysteine protease family

33. Proton gradient – a central interconvertible currency of free energy (Mito. inner membrane)

34. P:O ratio:  the number of molecules of inorganic phosphate incorporated into organic form per atom of oxygen consumed.  the number of molecules of ATP synthesized per pair of electrons carried through electron transport. ATP synthesis: is quantitative as phosphate uptake, conversion of orthophosphate to organic phosphates. Electron pairs: are quantitative as oxygen uptake. matrix NADH: 2.5 matrix FADH 2 : 1.5 + 2,4-dinitrophenol: P/O ratio from 2.5  0 Ex. 6

35. 96C 96T 192

36. 97T 97C

37. 98T 98C