Student Questions and Answers November 19, 2002

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1 Student Questions and Answers November 19, 2002 Q 1. Why is the D-glycerol phosphate shuttle used? The malate-aspartate shuttle is better, because there is no energy lost. If there is a problem with the concentration of oxaloacetate, the cell could easily generate more oxaloacetate. Q 2. If the shuttle transport does not depend on energy it would be a perpetuum mobile. How is this possible? Answer: FK: The malate-aspartate shuttle can function effectively only in cells in which the NADH/NAD+ ratio is much higher in the cytosol than in the mitochondrial matrix (a situation usually found in liver, kidney, and heart muscle cells). This simply represents a transporter-facilitated downhill transport of NADH. In the phospho glycerol shuttle part of the free energy is sacrificed to allow even an uphill transport: in brain and skeletal muscle mainly this shuttle is operative, since here usually NADH levels in the cytosol are considerably lower than within the mitochondria. Q 3. Are both shuttle mechanisms present in all cells? Is the malate-aspartate shuttle also used for other purposes, e.g. to transport amino-groups out? Answer: FK: (see above) The shuttle also serves to transport ammonia (from glutamate) from the mitochondria to the cytosol, which, among others, is fed into the urea cycle ( aspartate-argininosuccinate-shunt of the TCA cycle. Q 4. Why is the radical (semiquinone) form of ubiquinone not harmful to the cell? Answer: FK: To some extent it actually is. It is estimated that 1 to 2% of all electrons traveling down the mitochondrial respiratory chain form superoxide, and ubisemiquinone is either directly or indirectly responsible for >80% of them. This value is kept relatively) low by the various binding sites for CoQ in complex I and III, which significantly stabilise the radical semiquinone form, so that most of them will be fully reduced within their lifetime. Q 5. How long does CoQ stay a radical? Wouldn t it react very quickly with the other molecules around? Answer: FK: Unfortunately I couldn t find numbers. Otherwise see above. Q 6. When the oxidised ubiquinone is reduced to the free radical, the electron and the proton are added at different sites of the molecule and therefore forming the radical? And when the semiquinone is further reduced, this addition is the other way round addition of the electron where the proton was added first, and vice versa? Answer: FK: e - 2 H + Q 7. The E for complex I is larger than that for complex III. So are there more protons translocated by complex I than by complex III?

2 Answer: FK: Please see the answer to Q21, Nov 14; the detailed mechanisms of the coupling of electron transfer and proton translocation are not known for complex I and for complex IV, whereas for complex III, the Q-cycle already suggested by Mitchell has been experimentally proven. For both complexes different models have been proposed; most suggest the translocation of 4 protons (per 2 electrons) for complex I, and 2 protons for complex IV (and another 2 protons whih are consumed on the matrix-side by formation of H2O, but this is balanced with 2 protons, which are released to the matrix when the original substrate is oxidised). Q 8. How many ATP synthases are located in the membrane? (How many electron transport chains? How large is the distance between them?) Q 9. Is there a stoichiometry between the different complexes of the e-transport chain? Q 10. Is there the same number of electron transport chains and ATP synthase complexes in the membrane? Answer: FK: (Part of the answers provided in the lecture, see also Q17/Nov 14) In mammalian mitochondria, the stoichiometry of complex I : III : IV is about 1 : 3 : 6. It s not so easy to find numbers, but average figures are ~ 1500 copies of cytochrome oxidase (complex IV) and about 500 copies of F 1 -ATPase (complex V) per µm 2 ; (roughly the same number is given for the corresponding ATPase in chloroplasts, CF 1, while about 1500 complexes of PSI (photosystem I) were detected per µm 2 of the thylakoid membrane). Q 11. Charge of mitochondrial membrane: are these charges just in a small area directly above the membrane or are these charges distributed the whole space of the compartment? Q 12. Do the negative charges within the matrix accumulate along the membrane, or do they diffuse within the matrix, so there is auniform distribution of ions within this compartment? Answer: FK: Membrane potentials arise from unequal charge distributions in the solvent layer immediately adjacent to the membrane. You have to take into account that a membrane potential of ~50 mv means a voltage gradient of >100,000 V/cm (across the inner mitochondrial membrane it is even 300,000 V/cm!), so the respective charges have to sit immediately on the surface of the insulating bilayer. Q 13. Are there numbers of how many protons that have been pumped into the intermembrane space are used for 1) ATP synthesis, or b) symport of pyruvate, Pi, etc.? Answer: FK: Phosphorylation of ADP requires the passage of 3 protons through the F 1 /F 0 -ATPase, but the transport of ATP (out) and ADP + Pi (in) is driven by the proton gradient, which requires the equivalent of another proton per ATP. The total amount of pmf or ATP necessary to drive the various active transport systems depends, of course, of the metabolic situation of the cell, but can amount up to 2 / 3 of the proton gradient formed by electron transport. Q 14. Cytochrome c is associated to the membrane at the inter membrane space-side because of its positively charged sise chains. But the electrochemical potential of the membrane makes this side also positively charged? Answer: FK: (answer provided in the lecture)

3 Q 15. ATP synthase: does the shaft have a special structure that by it s rotation causes the conformational changes in the α and β-subunits? Answer: FK: The γ-subunit of the F0-rotor is asymmetric and induces conformational changes in the F1-part of the ATP synthase: crystal and NMR-structure studies demonstrate that α3β3 alone has perfect 3-fold symmetry, the ATPase activity follows Michaelis-Menten kinetics, and there is no cooperativity; if the γ-subunit is included in this molecule, one of the three β-subunits clearly shows a different conformation than the other two (whereas the conformation of the 3 α-subunits still is nearly identical) and now the high affinity nucleotide binding of one of the 3 sites can be observed. Q 16. Does the proton flow really work like shown in the movie? How are they really handed over? Answer: FK: Sorry, I don t remember exactly how the proton flow was suggested in the video sequence. Typical models generally follow this scenario: protons from the intermembrane space have access to the carboxylate group of the essential aspartate in the transmembrane helix of one of the c-subunits. Protonation of this side chains makes the interaction of this helix with a transmembrane helix of the a-subunit unfavourable, and the c-subunit is therefore displaced by rotation of the entire rotor complex (consisting of e.g. 12 c-subunits). At its new position the proton now can escape to the matrix. Q 17. ATP synthase: what is the function of the α-subunit? (asked twice) Answer: FK: A couple of residues of the α-subunits contribute to substrate binding. Very recently it was also shown that the N-terminus of α-subunits, located at the top of the F 1 hexamer is essential for energy coupling between proton translocation and catalytic activity. This domain appears to be connected to F 0 by OSCP (F 0 subunit conferring sensitivity of the complex to oligomycin), which in turn contacts the central rotary shaft. There are also reports about a heat-shock protection-like function of this subunit (showing some homology to hsp-60 s). Q 18. Are more copies of ATP synthase built into the membrane if more energy is required, or is the number of mitochondria increased? Answer: FK: Obviously the composition of the inner mitochondrial membrane remains largely unchanged under different metabolic conditions. However, under extreme conditions F 1 F 0 -ATP synthase may function in a different way! In the absence of oxygen the most important ATP-dependent process in animal cells is ion pumping by Na/K-ATPase to maintain the membrane potential, and thus membrane integrity. Under these conditions ATP synthase will operate as ATPase, pumping protons from the matrix, in order to maintain the mitochondrial membrane potential. This action is, however, limited by an inhibitory subunit of F 0 (IF1), which blocks the F 1 -part in a phdependent manner. As you suggested in your question, mitochondria are dynamic structures that alter their organization and metabolic capacity in response to changing environmental conditions. Yeast growing on a fermentable carbon source such as glucose has highly reticulated mitochondria comprising ~3% of the cell volume. This fraction increases up to 12% when grown on nonfermentable carbon sources (e.g. ethanol). When cells have entered

4 the stationary phase they show large numbers of separate, small mitochondrial compartments. Under conditions of starvation (or switching from a nonfermentable to a fermentable carbon source) most mitochondria are autophagised by the vacuole. Q 19. How conserved are the sequences of the subunits of ATP synthase in evolution? Answer: FK: The ATP synthases are conserved through evolution. One subfamily comprises the bacterial enzymes and those from mitochondria of animals, plants and fungi, as well as the chloroplast ATPase. There is a second family of closely related enzymes from Archaea, aslo comprising the H + -ATPase found in eukaryotic vacuoles. Surprisingly, the number of c-subunits of the rotary (F 0 ) unit differs between different systems: there are 12 copies in bacterial ATP synthase, 14 in CF 0 from chloroplasts of higher plants, but only 10 in the yeast enzyme. Accordingly, the stoichiometry of protons per ATP varies with system, and not necessarily is an integral number. Q 20. If you had other NDPs (like GDP) instead of ADP, could you synthesise GTP with the ATP synthase (maybe in vitro)? Answer: FK: The substrate specificity of F 1 is rather high; the order of binding strength is ADP GDP>IDP CDP. Q 21. When the concentration of ATP is too high, it is hydrolysed to ADP + Pi, while protons are pumped out. Is there any mechanism to prevent this? Wouldn t it be enough to just shut down ATP synthesis and wait until enough H + have been ejected via the electron transport chain? Answer: FK: Q 22. Is every catalytic centre of the ATP synthase filled with ADP + Pi, or could they also stay empty because no ADP + Pi reaches the centre in time for a new round? Or is a filled reaction centre necessary for rotation (the enzyme somehow recognises that one site is empty and waits until ADP and Pi have bound)? Answer: FK: In experiments with isolated F 1, at low ATP concentrations some of the binding sites remain empty, and the ATPase loses its subunit co-operativity, but the reaction still proceeds at a lower rate. Apparently, this cannot occur in vivo; proton flow requires substrates bound to F1 (a single-residue exchange in subunit b leads to uncoupling of proton flow and ATP synthesis in yeast). Recent studies suggest that the catalytic turnover of ATP synthase leads to a compression of the stator stalk; besides being a mechanism of energy conservation, this could be a condition for further rotation of the stalk. Anyway, due to the large affinity of F 1 for ADP+Pi, and ATP, respectively, empty active sites are very unlikely under normal substrate concentrations. Q 23. In the membrane potential the electrostatic interaction of the counterions acts over a distance of ~ 5 nm (the membrane). So why is it that interactions in polypeptides (e.g. between enzyme and substrate) need a very short distance (~ 0.5 nm) to occur? Answer: FK: The phospholipid bilayer acts as an insulator (low dielectricity constant), so the force attracting opposing charges are much larger than in solution (remember that the d.c. of water is abnormally high, so it is comparatively easy to separate charges in free solution).

5 Q 24. Where does an electrochemical gradient with the inside positively charged occur? Can it be useful for anything? Answer: FK: The electric potential built up by proton pumping across the thylakoid membrane has the opposite orientation: positive at the lumenal side, negative outside. The same is true with endosomes/lysosomes. AS far as cell membranes are concerned, I think the resting potential is always inside negative. Q 25. Which interactions of the rotating shaft with the α- and β-subunits cause the conformational changes (charged side chains, )? Answer: FK: Two of the β-subunits have almost identical conformation (β DP ), the third one (β E ) is clearly different (see Q15 above). In β DP there is strong hydrogen bonding between β-e381 and γ-r242, which is not possible for the β-subunit which faces the back of this part of the γ-subunit. This allows a conformational change of β E, which itself is stabilised by hydrogen bonding involving β-d302, T304, D305 and γ-r268, Q269, and T273 (all numbering for F 1 F 0 -ATP synthase from E. coli). Q 26. Doesn t the rotation of the shaft create a kind of tension in the membrane? Answer: FK: This should not lead to any tension, since membrane proteins and the phospholipid bilayer can move independently. Moving trans-membrane helices within the membrane certainly will require some energy (a number of weak interactions are broken and presumably the same number is formed, but there must be some activation barriere), but this should not be significant at ordinary temperatures. Q 27. Does complex V/ATPase age i.e. does its performance decline with time? Answer: FK: As with all other proteins, there will be some modifications with time (mostly oxidative), some of them detrimental for proper functioning. Unfortunately I can t give you any information about in vivo-turnover of the components of this complex, which you would have to know to judge whether this could be a significant problem or not. Q 28. You mentioned that yeast cells may live without functional electron transport chain. This means that they have to rely on passive transport (diffusion), right? Answer: FK: These mutants ( petite strains ) effectively live anaerobic irrespective of the availability of oxygen, and thus can only grow on fermententable carbon sources. The required amount of ATP will, of course, be spent for active transport. Q 29. How does ADP + Pi bind to the O-side of the ATPase? Answer: FK: The term O does not mean that this site does not bind the reactants at all, it only shows significantly lower binding affinity as compared with state L (see table below). Substrate binding induces the conformational change leading to state L, which by co-operativity facilitates tight substrate binding (state T) in the neighbouring subunit. The rotating shaft then promotes release of the product and state O is re-established.

6 Binding strength Kd (µm) T L O Mg.ATP Mg.ADP Mg.Pi 10,000 > 10,000 > 10,000