1. Mitochondria and chloroplasts SBS922 Membrane Biochemistry John F. Allen School of Biological and Chemical Sciences, Queen Mary, University of London 1

2. http://jfa.bio.qmul.ac.uk/lectures/

3. School of Biological and Chemical Sciences Seminars 2006-07 WEDNESDAYS AT 12 NOON IN LECTURE THEATRE G23, FOGG BUILDING, SCHOOL OF BIOLOGICAL AND CHEMICAL SCIENCES 6 December 2006PROFESSOR SO IWATA David Blow Chair of Biophysics and Director of Centre for Structural Biology Division of Molecular Biosciences, Imperial College London, and Diamond Light Source, Rutherford Appleton Laboratory, Chilton Structural studies on membrane proteins 28 February 2007Professor COLIN ROBINSON Department of Biological Sciences, University of Warwick, Coventry Pathways for the targeting of proteins across chloroplast and bacterial membranes

4. The membrane energised state We have seen how observed characteristics of oxidative phosphorylation led to conclusion that there was a membrane energised state linking electron transfer in mitochondria to ATP synthesis and other membrane-linked energy-dependent functions such as active transport of solutes.

5. There was convincing evidence by 1960 that the transfer of energy via this membrane energised state between the respiratory electron transfer chain, the synthesis of ATP and the various solute transfer systems is both efficient and fully reversible.

6. Membrane Energised State (cont.) It was occurring via a common (to all these activities) and stable non- phosphorylated energised state. This energised state was dissipated by uncoupling agents (uncouplers), which led to a permanent increase in the rate of electron transfer. The energised state was not dissipated by phosphorylation inhibitors, which caused a decrease in the rate of electron transfer which could be overcome by uncouplers.

7. Thus respiration will drive ATP synthesis, ATP hydrolysis will drive reversed electron transfer, and both respiration and ATP hydrolysis will drive solute transport. Similarly certain solute gradients of the correct magnitude and direction will power both ATP synthesis and reversed electron transfer. It was clear therefore that the energised state occupies a central position in the mechanism of membrane-associated energy transduction.

8. This energised state was essentially linking together two types of protein complex in coupling membranes. a) electron transfer complexes b) ATP synthase Three types of coupling membranes are the IMM, thylakoid membrane and plasma membrane of prokaryotes, and all used same energised state. Search for the nature of the energised state became one of central problems in Biochemistry. Only precedent was the phophorylated intermediate in substrate-level phosphorylation, but energised state of coupling membranes was non-phosphorylated, and an intact membrane was required to couple electron transfer to ATP synthesis.

9. CHEMIOSMOTIC HYPOTHESIS Proposed by Peter Mitchell 1961 and further elaborated 1966 Competing hypotheses 1) Chemical hypothesis (i.e. like substrate-level phosphorylation) 2) Conformational hypothesis 3) Localised proton hypothesis (variation of chemiosmotic) Peter Mitchell awarded Nobel Prize in Chemistry in 1978.

11. Mitchell proposed that electron transfer directly produced an electrochemical gradient of protons across the coupling membrane that was subsequently used to drive ATP synthesis. The theory was subsequently adapted and expanded, principally by Mitchell and his colleague Jennifer Moyle to account for other membrane-linked energy dependent functions such as the active transport of solutes across the membrane. The chemiosmotic hypothesis is named because it is postulated to involve both a) chemical reactions, the transfer of chemical groups (electrons,protons and O 2 2- ) within the membrane b) osmotic reactions, the transport of a solute (protons) across the membrane

12. Transmembrane Proton gradient Energy transduction occurs via a proton circuit which circulates through the insulating coupling membrane and the two adjacent bulk phases ( the matrix and the cytosol/ intermembrane space in mitochondria). Since each of the two bulk phases is in equilibrium, energy storage is transmembrane rather than intramembrane (intramembrane proton gradients were the basis of the localised proton hypothesis).

13. This energy storage takes the form of a delocalised electrochemical potential difference of protons (  H+ ), otherwise known as the protonmotive force (p.m.f. or  p). It is an electrochemical gradient because it is composed of both A chemical potential difference  pH ( pH out -pH in ) An electrical potential difference or membrane potential  These two contribute to  p according to the following relationship  p =  - Z  pH where Z= 2.303RT F R= gas constant, T= absolute temperature in Kelvin, F= Faraday Z constant approximately equal to 60 at 25 o C and serves to convert pH into electrical units, mV.

14. ESSENTIAL REQUIREMENTS OF THE CHEMIOSMOTIC HYPOTHESIS MITHCHELL PROPOSED THREE ESSENTIAL REQUIREMENTS THAT HAD TO BE VERIFIED EXPERIMENTALLY BEFORE THE CHEMIOSMOTIC HYPOTHESIS COULD BE ACCEPTED AS PROVEN.

15. Mitchell’s three essential requirements 1) That the respiratory chain redox system (electron transfer) translocates protons across the membrane in one direction (anisotropic,direction-oriented) as electrons flow down the chain. 2) The coupling membrane should be impermeable to protons and other ions except via specific exchange-diffusion systems which are involved in active solute transport 3) That the ATP synthase can transport protons across the membrane in one direction down the concentration (  pH ) and charge (  ) gradient, using the energy for ATP synthesis. Alternatively it should be able to use the energy from ATP hydrolysis to pump protons in the opposite direction (active transport against the concentration and charge gradient).

16. Only central part of Mitchell’s hypothesis accepted, that an electrochemical gradient of protons was both necessary and sufficient for ATP synthesis linked to electron transfer. Specific mechanisms he proposed for proton pumping were only partially correct, and his chemiosmotic mechanism for ATP synthase was wrong (this actually involves conformational change in ATP synthase caused by p.m.f., and was partly elucidated as a result of part of the structure of the ATP synthase being solved by John Walker at Cambridge, who also received the Nobel Prize).

17. Was convincing evidence by 1960 that the transfer of energy via this membrane energised state between the respiratory electron transfer chain, the synthesis of ATP and the various solute transfer systems is both efficient and fully reversible.

18. Transmembrane Proton gradient Energy transduction occurs via a proton circuit which circulates through the insulating coupling membrane and the two adjacent bulk phases ( the matrix and the cytosol/ intermembrane space in mitochondria). Since each of the two bulk phases is in equilibrium, energy storage is transmembrane rather than intramembrane (intramembrane proton gradients were the basis of the localised proton hypothesis).

19. This energy storage takes the form of a delocalised electrochemical potential difference of protons (  H+ ), otherwise known as the protonmotive force (p.m.f. or  p). It is an electrochemical gradient because it is composed of both A chemical potential difference  pH ( pH out -pH in ) An electrical potential difference or membrane potential  These two contribute to  p according to the following relationship  p =  - Z  pH where Z= 2.303RT F R= gas constant, T= absolute temperature in Kelvin, F= Faraday Z constant approximately equal to 60 at 25 o C and serves to convert pH into electrical units, mV.

20. Mitchell’s three essential requirements 1) That the respiratory chain redox system (electron transfer) translocates protons across the membrane in one direction (anisotropic,direction-oriented) as electrons flow down the chain. 2) The coupling membrane should be impermeable to protons and other ions except via specific exchange-diffusion systems which are involved in active solute transport 3) That the ATP synthase can transport protons across the membrane in one direction down the concentration (  pH ) and charge (  ) gradient, using the energy for ATP synthesis. Alternatively it should be able to use the energy from ATP hydrolysis to pump protons in the opposite direction (active transport against the concentration and charge gradient).

21. ATP synthase can transport protons across the membrane in one direction down the concentration (  pH ) and charge (  ) gradient, using the energy for ATP synthesis.

22. Alternatively the ATP synthase should be able to use the energy from ATP hydrolysis to pump protons in the opposite direction (active transport against the concentration and charge gradient).

23. Only central part of Mitchell’s hypothesis accepted, that an electrochemical gradient of protons was both neccesary and sufficient for ATP synthesis linked to electron transfer. Specific mechanisms he proposed for proton pumping were only partially correct, and his chemiosmotic mechanism for ATP synthase was wrong (this actually involves conformational change in ATP synthase caused by p.m.f., and was partly elucidated as a result of part of the structure of the ATP synthase being solved by John Walker at Cambridge, who also received the Nobel Prize).

25. EVIDENCE FOR FIRST REQUIREMENT 1. That the respiratory chain redox system (electron transfer) translocates protons across the membrane in one direction (anisotropic,direction-oriented) as electrons flow down the chain. i.e "An anisotropic proton- translocating respiratory electron transfer chain" YOU CAN ONLY MEASURE THE INITIAL EJECTION OF PROTONS FROM MITOCHONDRIA AS ELECTRON TRANSFER STARTS, BEFORE THE RE-ENTRY OF PROTONS HAS BECOME ESTABLISHED. BY DEFINITION DURING STEADY STATE ELECTRON TRANSFER THE RATE AT WHICH PROTONS ARE PUMPED OUT OF MITOCHONDRIA EQUALS THE RATE OF THEIR RE-ENTRY.

26. Precautions )incubate mitochondria anaerobically so no electron transfer )in lightly-buffered medium so pH/[H + ] changes can be observed )add oligomycin to inhibit proton re-entry via the ATP synthase )add valinomycin ( a potassium ionophore ) and a high concentration of KCl to abolish  and thus maximise  pH.

27. )add a small non-saturating pulse of oxygen to start electron transfer and measure the extent of proton extrusion into the medium surrounding the mitochondria.

28. RESULTS

29. FACTORS CONTRIBUTING TO DECAY OF  pH Protons decay back in to mitochondria across IMM because of 1] inherent proton permeability of IMM – note that FCCP accelerates the decay 2] action of endogeneous Na + /H + antiport 3] electroneutral PO 4 entry

30. EVIDENCE FOR SECOND REQUIREMENT 2) The coupling membrane should be impermeable to protons and other ions except via specific exchange-diffusion systems which are involved in active solute transport. It can be deduced that the inner mitochondrial membrane has a low effective proton conductance by studying the action of uncouplers. A majority of uncouplers act by increasing the effective proton conductance of the coupling membrane, dissipating the proton motive force and thus breaking the link between electron transfer and ATP synthesis. It is reasonable to assume that the coupling membrane has a low proton conductance in their absence.

31. MECHANISM OF ACTION OF UNCOUPLERS (i.e. FCCP) Uncouplers are lipophilic (membrane-permeable) weak acids that can cross the membrane in either the protonated or deprotonated form. So they act as proton translocators, catalysing a proton uniport across the coupling membrane.

32. Driven by  pH Driven by 

33. Subsequently Mithchell and Moyle measured the effective proton conductance of the inner mitochondrial membrane. This effective proton conductance is one million times less than that of the surrounding aqueous phases. i.e. C m H + < or = 0.5µmho cm -2 Or 0.2 nmol H + min -1 mg protein -1 mV  p -1

34. EVIDENCE FOR THIRD REQUIREMENT 3) That the ATP synthase can transport protons across the membrane in one direction down the concentration (  pH ) and charge (  ) gradient, using the energy for ATP synthesis. Alternatively it should be able to use the energy from ATP hydrolysis to pump protons in the opposite direction (active transport against the concentration and charge gradient) i.e. "A reversible proton-translocating ATP synthase"

35. EVIDENCE FOR THIRD REQUIREMENT (CONT.) a)Mitchell and Moyle showed that if you injected a small amount of ATP into a suspension of anaerobic ( so no electron transfer) mitochondria there was an expulsion of protons as ATP was hydrolysed.

36. b)In mitochondria need to impose a artificial  across the inner mitochondrial membrane, as active solute transport uses up  pH, so mitochondria work on  for ATP synthesis. Finally achieved in early 1970's.

37. EVIDENCE FOR THIRD REQUIREMENT (CONT.) First demonstration of artificial  pH driving ATP synthesis was in 1966 by Jagendorf and Uribe, using thylakoid membranes of chloroplasts which don't have solute transport and normally work on high  pH and low . [see handout]

38. EVIDENCE FOR THIRD REQUIREMENT (CONT.) First demonstration of artificial  pH driving ATP synthesis was in 1966 by Jagendorf and Uribe, using thylakoid membranes of chloroplasts which don't have solute transport and normally work on high  pH and low . [see handout]

39. OTHER EVIDENCE FOR MITCHELL’S CHEMIOSMOTIC HYPOTHESIS