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BIOENERGETICS: OXIDATIVE PHOSPORYLATION Student Edition 10/23/14 Version Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall

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Presentation on theme: "BIOENERGETICS: OXIDATIVE PHOSPORYLATION Student Edition 10/23/14 Version Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall"— Presentation transcript:

1 BIOENERGETICS: OXIDATIVE PHOSPORYLATION Student Edition 10/23/14 Version Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 213 Maddox Hall Web Site: Original material only © B. Chazotte

2 Goals Learn the basic tenets of the chemiosmotic hypothesis and how mitochondrial structure is consistent with the hypothesis. Consider how thermodynamics describes electrochemical gradient & oxidative phosphorylation. Learn the quantitative description of the proton electrochemical gradient and its components. Be able to calculate the phosphorylation potential. Be able to calculate the free energy of hydrolysis. Learn the basic components involved in pumping proton across the mitochondrial inner membrane. Understand the concept and meaning of the P:O ratio. Learn the structure of the F 1 F o -ATPase and how Boyer’s binding exchange involves this structure in the synthesis of ATP. Understand how fluorescent dyes can report on the (mitochondrial) membrane potential in living cells and how changes in the potential due to substrates and inhibitors can be monitored. Understand how gradients involve the use or storage of energy.

3 CHEMIOSMOTIC HYPOTHESIS Tenets 1.Energy transducing membranes are vesicular, sealed, and impermeable to protons except for the pathways involved in redox mediated or protein-catalyzed H+ translocation. 2.Energy is stored in a pH gradient or membrane potential which are energetically equivalent, with the electrochemical potential. 3. The    is formed by vectorially alternating H + and e - carriers in the electron transport chain. 4.An H + flux is coupled to the ATP synthase/ATPase catalyzed by the large multisubunit F 0 F 1 ATPase. Each reaction is anisotropic with respect to this flux. The synthesis reaction is coupled to an H+ flux driven by the  H+ from the  H+ –positive side of the membrane. 5.Uncouplers of energy transduction were predicted to be lipid-soluble weak acids or bases that can catalyze the equilibration of H+ of OH- across the membrane. Cramer & Knaff 1990

4 THERMODYNAMICS Brief Description Chemical Work and Chemical Potential The chemical potential  i, of compound I, is the free energy per mole   G   n i  T,,p,,n j  n i     is the partial derivative with respect to n i when temperature, pressure, and other n are held constant.  is important for transport problems because it is the change in free energy of a system per mole of component moved in or out of the system. Cramer & Knaff 1990

5 Proton Electrochemical Gradient (  H+ ) #1 The electrochemical difference of protons across the mitochondrial inner membrane provides the high energy state to drive ATP synthesis, ion and substrate transport, transhydrogenation, and other energy requiring reactions. One can write for hydrogen transport across, e.g., the mitochondrial inner membrane, where F= Faraday constant and  membrane potential in mV  H+ = F  – RT log (H i + /H o + )  H+ = F  – RT  pH  H+ is composed of two components: a membrane potential (charge difference; electrical potential):  a pH gradient (concentration difference; chemical potential):  pH In mitochondria  is the bulk of the contribution to  H+

6 Components of Mitochondrial Proton Gradient Topic:OxPhos Alberts et al Fig 14-19

7 Proton Electrochemical Gradient (  H+ ) #2  H+ = F  RT  pH F =Faraday constant = 96,487 coulombs/mole = 96.5 kJ mol -1 V -1 = kcal mol -1 V -1 R = gas constant = J deg-1 mol-1 = cal deg-1 mol-1 = liter atm deg-1 mol-1 T = absolute temperature Represents the free energy change in kJ/mole when 1 mole of H + moves into the mitochondrion. Expressing the proton electrochemical gradient in millivolts is called the phosphorylation potential (  p).  p=  H+ / F =  (RT/F)  pH  is the bulk of the contribution to  H+

8 Determination of  pH Calculate from equilibrium distribution of weak bases. Use Henderson-Hasselbalch equation [HA] in = [HA] equilibrium K a = ([H + ] in [A - ] in )/[HA] in = ([H + ] out [A - ] out )/[HA] out  pH = log([A - ] in / [A - ] out ) Note: mitochondrial  pH is typically less than 1 pH unit

9 Calculation using Phosphorylation Potential  p Problem: Calculate the pH gradient at 37 ºC required across the mitochondrial inner membrane to equal a membrane potential of -150 mV. The relevant equation for the phosphorylation potential in mitochondria, which is in mV units already, is:  p =  H+ / F =  (RT/F)  pH  = -150 mV ( J deg -1 mol -1 * ºK/96.5 kJ mol -1 V -1 )  pH  = -150 mV ( J deg -1 mol -1 * ºK/96,500 J mol -1 V -1 )  pH 0.15  V  = (2.672*10 -2 V -1 )  pH 0.15  V  = V -1  pH  pH = -2.44

10 Proton Motive Force in Oxidative Phosphorylation Horton et al 2012 Figure 14.9

11 Uses of the Proton Gradient Berg, Tymoczko, & Stryer 2012 Fig 18.44

12 PROTON PUMPING & OXIDATIVE PHOSPHORYLAYION

13 Schematic of Electron Transport Enzyme Complexes

14 H + ions transported across a membrane per unit area to generate  = 100 mV  = Q/C where C is the specific membrane capacitance. Q is the charge per unit area. For biological membranes C ~ 1  farad/cm 2. Thus, if  = 0.1V and C= coulombs/cm 2, Q = coulombs/cm 2. Charge on one proton = coulombs # protons translocated per unit area = 6 x /cm 2. # protons translocated per sq micron = 6 x10 3 For a 300 Å diameter vesicle the translocation of 20 protons would generate a 100 mV potential. For a typical rat liver mitochondrion estimate: 6 x protons /cm 2  cm 2 /mg protein  8.7 x 10 9 mitochondria /mg protein = 35,903 protons/mitochondrion Cramer and Knaff 1990

15 “THE” CHEMIOSMOTIC EXPERIMENT Berg, Tymoczko, & Stryer 2012 Fig 18.23

16 Factors Controlling the Partition of  p Components   and  pH Nicholls & Ferguson Bioenergetics

17 Proton Pumping in Electron Transport Lehninger 2000 Fig 19-15

18 Lehninger 2000 Fig Topic:Electron Transport OXPHOS OVERVIEW

19 Lehninger 2000 Fig Q Cycle Schematic Voet, Voet, & Pratt 2013 Fig 18.15

20 Control of Oxidative Phosphorylation

21 P: O Ratios Revisited in the Chemiosmotic World P:O, ATP:O, ATP/2e-, 2H+/e- ! The ratio of electrons transported to hydrogen ions pumped is an important number in oxidative phosphorylation. It is generally agreed now that FOUR protons are consumed to produced 1 ATP. One of those protons is used in transporting ATP, ADP and Pi.

22 P:O Ratios in Electron Transport Voet, Voet & Pratt 2006 pp ATP synthesis is tightly coupled to the proton gradient. Possible to express the amount of ATP synthesized in terms of the substrate molecules oxidized. Experiments had shown approximately 2.5, 1.5, and 0.5 ATP synthesized with oxidations of NADH (via complex I), FADH 2 (via complex II) and TMPD (via Complex IV, artificial 2e- donor). P:O ratio relates amount of ATP synthesized to amount oxygen reduced. Years of controversy over ratios. Integer or non-integer. Likely non- integer. Chemiosmotic hypothesis unlike other theories does not need whole numbers.

23 MitochondriaRedoxStates Mitochondria Redox States (according to Chance and Williams [Adv. Enz ]) [O 2 ]ADPSubstrateRespiration Limiting State 1 >0lowlowslowADP State 2 >0high~lowslowsubstrate State 3 >0highhighfaste - trans State 4 >0lowhighslowADP State 5 0highhigh0oxygen Oxidative phosphorylation is occurring during state 3 respiration

24 Polarographic Determination of P/O Ratio State 1 State 4 State 3 State 5

25 COMPONENTS INVOLVED IN OXIDATIVE PHOSPHORYLATION [DIRECTLY & INDIRECTLY]

26 F 1 F 0 ATP Synthase Polypeptide Structure Ref:Sarasate Fig 5 Science Voet, Voet, & Pratt 2013 Fig (inverted)

27 OXIDATIVE PHOSPHORYLATION: ATP Synthase Binding Model Topic: OxPos

28 Lehninger 2000 Fig Topic:Ox Phos ATP Synthase Binding Site Model Voet, Voet & Pratt 2008 Figure O - catalytically inactive & very low ligand affinity L – catalytically inactive & loose ligand binding T – catalytically active & tight ligand binding 1)ADP & P i bind to site “L” 2)“L” converted to “T” site by energy driven conformational change 3)ATP is synthesized at site “T” and release as “T” becomes “O” site during energy driven conformational change 3-αβ “subunit pairs” in F 1. β binds nucleotide

29 ATP Synthase (F 1 F 0 ) Structure Voet, Voet & Pratt 2002 Figure Negative Stain EM Cryo EM (F 1 F 0 E. Coli) Artist Illustration

30 Voet, Voet & Pratt 2013 Fig Topic:Ox Phos Proof of ATP Binding Model FoFo F1F1

31 OXIDATIVE PHOSPHORYLATION ADP/ATP Translocator 1 Horton et al 2012 Fig Topic: OxPhos

32 ADP-ATP Translocator: Conformational Mechanism Voet, Voet & Pratt 2008 Figure 18.6

33 Lehninger 2000 Fig Topic:Electron Transport OXPHOS TRANSPORTER RELATIONSHIPS

34 e - Transport & Oxidative Phosphorylation Lehninger 2000 Understand Biochemistry CD

35 MITOCHONDRIAL OxPhos Free Energy of Hydrolysis in a Cell Lehninger 2000 Table 14-5 Topic:OxPhos  G°´= –30.5 kJ/mol for ATP. However, that is based on standard conditions, i.e. 1 molar. pH 7.0, which may not be the conditions in a living cell. Consider a human erythrocyte

36 Free Energy of Hydrolysis in a Cell. II Lehninger 2000 Box 14-2 Topic:OxPhos [ADP] 1 [P i ] 1  G p =  G  ´ RT log K´ eq =  G  ´ RT log [ATP] 1 [2.50 x ] 1 [1.65 x ] 1 =  J mol – x J mol –1 K -1 x 298  K * log [2.25 x ] 1 =  J mol –1 + (5,707 J mol –1 x ) =  J mol –1 – 21,327 J mol –1 = - 51,827 J mol –1 for hydrolysis and 51,827 J mol –1 for ATP synthesis

37 Nernst Equation  = -(RT/F) ln(A in / A out ) Which at room temperature simplifies to  = -59  ln(C in / C out )  = the membrane potential A x = probe chemical activity inside or outside R = gas constant T = absolute temperature F = Faraday constant C x = the probe concentration inside or outside

38 Calculation using the Nernst Equation Given: TMRM concentration = 50 mM inside and 5 nM outside the mitochondrion at 37  C  = - (RT/F) ln(C in / C out )  = - ( J mol –1  K -1 x 310  K / 96,500 J mol -1 V -1 ) ln(50 µM in / 5nM out )  = - ( J mol –1  K -1 x 310  K / 96,500 J mol -1 V -1 ) ln(10,000 )  = - ( 2, J mol –1  / 96,500 J mol -1 V -1 )  = - ( V ) = V = mV

39 6AP16076 Nucleus Graylevel Image Confocal Image of Human Fibroblast Labeled with TMRM Cytoplasm Mitochondrion Pseudocolored Image

40 6AP02123 ROI PlateletMitochondria Mononuclear Leukocyte  based Pseudocolored Image Graylevel Image Selecting a Region of Interest to Histogram Human Mononuclear Leukocyte

41 Selected Inhibitors of Mitochondrial Bioenergetics CCCPcollapses  pH and  Valinomycincollapses  Rotenoneinhibits Complex I electron transport. Antimycin ainhibits electron transport at Complex III TTFAinhibits Complex II electron transport. KCNinhibits electron transport at Complex IV Oligomycinprevents ATP synthesis, increases  2-Deoxyglucosecauses mitochondrial respiratory jump

42 6MA MA13088CCCP CCCP Effects

43 HMINH1 Effects of Mitochondrial Inhibitors on IPDs of Human Mononuclear Leukocytes 2.8  M, 29  g/ml, 0.87  M

44 BIOENERGETICS OF CELLULAR TRANSPORT BIOENERGETICS OF CELLULAR TRANSPORT Topic: Bioeneregtics Transport

45 Thermodynamics of Ion Gradient For protons we have written:  H+ =  o +zF  RT log (H+) Likewise for a electrochemical sodium gradient we can write  Na+ = zF  RT log (Na + final ) (Na + initial )  = the membrane potential, R = gas constant, T = absolute temperature, F = Faraday constant, z = charge (for proton: z = +1) Cramer & Knaff 1990 pp18-19

46 Lehninger 2000 Fig 12-29

47 Active Transport Processes Driven via the Mitochondrial Gradient Topic:OxPhos Alberts et al Fig 7-21

48 Thermodynamics of  H+ –Linked Active Transport Symport If all of the free energy available in the  H+ is stored in the electrochemical potential, then we can write for  s of substrate accumulation in a symport mechanism. Where S refers to a solute molecule and n protons to transport one solute molecule  G total = n*  H +  s = 0 eq 1  H+ = F  RT log (H i + /H o + )eq 2  s = zF  RT log (S i +z /S o +z )eq 3 Where “ i ” is inside and “ o ” outside & for solute the initial state is outside and the final state is inside  i  o ; then Substituting eqs 2 & 3 into eq 1 0= zF  RT log (S i +z /S o +z ) + F  nRT log (H i + /H o + ) Divide by 2.303RT and Rearrange log (S i +z /S o +z ) = n  pH – (n+ z) F (  z) Cramer & Knaff 1990, pp 19-20

49 Thermodynamics of  H+ –Linked Active Transport Antiport If all of the free energy available in the  H + is stored on the electrochemical potential then we can write for  s of solute accumulation in an antiport mechanism. Where S refers to a solute molecule and n protons to transport one solute molecule  H+ = F  RT log (H i + /H o + )eq2 In antiport initial and finale states are opposite of symport so the terms in the log expression for solute are inverted:  s = zF  RT log (S o +z /S i +z )eq3 Where i is inside and o outside; then Substituting eqs 2 & 3 into eq 1 0= zF  RT log (S o +z /S i +z ) + F  nRT log (H i + /H o + ) Rearrange and Divide by 2.303RT log (S i +z /S o +z ) = - n  pH + (n - z) F (  z) Cramer & Knaff 1990, pp20-21

50 Lehninger 2000 Fig SUMMARY OF TRANSPORT PROCESSES

51 End of Lectures


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