Class Note for BIOC 460 at UA
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Date Created: 02/06/15
Bioc 460 Dr Miesfeld Fall 2008 Lecture 28 Oxidative Phosphorylation Magnetic bead Key Concepts STRUCTURE AND FUNCTION OF THE ATP SYNTHASE COMPLEX TRANSPORT SYSTEMS IN THE MITOCHONDRIA E TS AND OxPHOS ARE FUNCTIONALL YLINKED mnmcx Sirepiawdin 1 KEY CONCEPT QUESTIONS IN OXIDA TIVE PHOSPHORYLA TlON 395 39 How does proton motive force drive ATP synthesis Why does glucose oxidation in muscle cells produce two less ATP than in liver cells Explain why oligomycin inhibits succinate oxidation in isolated mitochondria Biochemical Application of Oxidative Phosphorylation N07 The uncoupling protein UCP1 controls thermogenesis in brown adipose tissue by providing a bypass route for protons to reenter the mitochondrial matrix without flowing through the ATP synthase complex resulting in the conversion of redox energy to heat Dinitrophenol is a toxic chemical uncoupler that was once used as a diet aid 3i i LIIDinitrophenol DN P Structure and Function of the ATP Synthase Complex The mitochondrial ATP synthase complex uses the protonmotive force generated via the electron transport system to synthesize ATP through protein Figure 1 conformational changes in a process called oxidative 39 Carbohydrate phosphorylation figure 1 In addition to generating ATP Metabolism during aerobic respiration a similar ATP synthase complex synthesizes ATP in response to proton motive generated by lightdriven photosynthetic processes in plant chloroplasts When Mitchell proposed the chemiosmotic theory there was already evidence that a large protein complex in the inner mitochondrial membrane was responsible for Amino Acid ATP synthesis Originally called complex V and later Men wa purified as an ATP hydrolyzing enzyme using a cellfree assay system the ATP synthase complex is now known i to represent one ofthe quintessential protein machines found in living cells As shown in figure 2 the K mitochondrial ATP synthase complex consists of two large FADH2 i NADH Metabolism Citrate Cycle i Carbon LL structural components called F1 which encodes the Electron l mamquot catalytic activity and F0 which functions as the proton gme quotWSWquot g Ph t th channel crossing the inner mitochondrial membrane The Phosphorylation 039 es39s subscript quotoquot in F0 written here in lower case to avoid 02 confUSIon With zero quot0quot refers to the finding that the ATP H20 antibiotic gligomycin inhibits the activity of the Fo proton channel The E coli F1 component consists of three 0 subunits three 3 subunits and one each of y 6 and 5 subunits denoted a3 3y s The isolated Fo component contains one a subunit two b subunits and 912 c subunits abgcg42 The discrepancy in the number of c subunits is due to the difficulty in purifying an intact E coli Fo structure from membrane fractions however recent studies suggest that the E coli Fo component is likely to 1 of11 pages consist of 10 0 subunits Figure 1 shows a model depicting the subunit arrangement of the F1 and F0 components of the E coli ATP synthase complex Although the F1 and F0 components represent the largest biochemical entities identified by protein purification it is useful to organize the F1Fo ATP synthase complex into three functional units in order to visualize the working parts of this protein machine as illustrated in figure 3 These three functional units can be described as 1 the rotor made up Ofy and 3 subunits which sit on top of the 0 subunit ring that rotates as protons enter and exit the ring 2 the catalytic head piece containing the hexameric x363 unit that contains an enzyme active site in each of the three 3 subunits and is responsible for ATP synthesis in the intact complex or ATP hydrolysis in the isolated F1 component and 3 the stator consisting of the a subunit imbedded in the membrane which contains two half channels for protons to enter and exit the F0 component and a stabilizing arm made up of the b dimer and 6 subunits a b26 The term stator refers to immobile parts of a rotary engine which describes the role of the 56 stabilizing arm in preventing the x363 headpiece from rotating along with the y subunit Structural studies of the ATP synthase catalytic head piece in the presence of ATP and ADP have revealed that the 3 subunit undergoes conformational changes depending on which nucleotide ATP or ADP is bound in the enzyme active site This discovery combined with enzyme kinetic analysis of the F1Fo ATP Figure 3 synthase complex provided essential clues to understanding the molecular basis of proton driven ATP synthesis Figure 4 shows a snapshot of two views of a model ATP synthase b2 complex that has been animated Bioc 460 Dr Miesfeld Fall 2008 2 of 11 pages catalytic activity of the three 6 subunits was regulated by conformational changes induced by the rotating y subunit provided the key to understanding the enzyme mechanism of the F1FO ATP synthase complex Nucleotide binding studies revealed that it was the affinity of the 3 subunit for ATP not the rate of ATP synthesis or ATP hydrolysis in isolated F1 fragments that was altered by proton ow through the F0 component This conclusion came from studies showing that in the absence of protonmotive force the dissociation constant KB of the F1 headpiece for ATP was about 103912 M however when the electrochemical proton gradient was supplied the kD dropped to 10396 M a decrease in the KB of a million fold In addition radioactive Bioc 460 Dr Miesfeld Fall 2008 The realization that the Figure 5 exchange experiments using purified F1 and 18O isotopicallylabelled Pi showed that the ATP ltgt ADP Pi reaction was in fact readily reversible within the enzyme active site confirming that protonmotive force was required forthe release of ATP not its formation Based on these results and on what was known about the subunit composition of the F1 component Paul Boyer at UCLA proposed the binding change mechanism of ATP synthesis to explain how conformational changes in 6 subunits control ATP production figure 5 The binding change mechanism incorporates four basic principles 1 The y subunit directly contacts all three 6 subunits however each of these interactions are distinct giving rise to three different 3 subunit conformations The ATP binding affinities of the three 6 subunit conformations are defined as T tight L loose and 0 open in which ADP and Pi bind to the O and L conformations and ATP binds tightly to the T conformation but is released from the enzyme when the 6 subunit is in the O conformation As protons flow through FO the y subunit rotates in a counterclockwise circle looking at F1 from the matrix side such that with each 120 rotation the 6 subunits sequentially undergo a conformational change from L gt Tgt O gt L The binding change mechanism model predicts that one full rotation ofthe y subunit should generate 3 ATP and require the translocation of 9 H across the membrane 3 of 11 pages Bloc 460 Dr Miesfeld Fall 2008 The binding change mechanism model predicts that the 5 subunit conformations should be interdependent such that when one subunit is in the T conformation the other two subunits must be in the O and L conformation With the input of sufficient protonmotive force 3 Hp flowing through F0 to become 3 HN the rotorturns 120 in the counterclockwise direction looking at F1 from the matrix side causing the asymmetricaly subunit to form the alternate conformation with each of the 5 subunits following the sequence L gt T gt O gt L This same sequence of events repeats two more times until the y subunit has completed a 360 rotation to produce 3 ATP as a result of 9 Hp flowing down the electrochemical proton gradient through F0 to become 9 HM Note that the ratio of 3 HATP is not yet certain because there are unanswered questions regarding the molecular mechanism of the protondriven rotor see below Nevertheless we will use 3 HATP here because it is consistent with the ATP currency exchange ratio of 25 ATPNADH as well as the proton pumping ratio of 10 HNADH by the electron transport system both of which have been empirically determined Since biochemical studies had shown that H flow through F0 is not directly involved in the catalytic mechanism and moreover that the F1 headpiece functions as an ATPase in the absence of F0 Boyer39s model predicts that ATP hydrolysis by the F1 headpiece should reversethe direction of the y subunit rotor To test this idea Masamitsu Yoshida 39M and Kasuhiko Kinosita of Tokyo Institute of Technology used recombinant DNA methods to modify the a 5 and y subunits of the E coli F1 component in order to build a synthetic molecular motor As shown in figure 6 they use an actin quotpropellerquot in place of the c subunit ring Intermembrane Space side Whlch allowed them to ATP synthesis U Clockwise monitor rotation of the y ATP I C I subunit using fluorescence hydro ys39s U Gunter COCkW39se microscopy When reaction buffer containing Mg2 and ATP was added to the slide it was observed that the actin propeller rotated in the clockwise direction viewed from the bottom looking up on the actin filament through the glass slide exactly as predicted In fact it wasn39t a smooth rotation but rather a ratchet motion consisting of 39 three 120 rotations for each complete circle a finding C 4 that was consistent with Boyer39s binding change mechanism The same research group later went on to build an ATP generating motor by attaching a magnetic bead to the actin filament and forcing the g subunit to rotate in the opposite direction counterclockwise when L i viewed from the same angle using a series of A A electromagnets figure 7 Clockwise counterclockwise matrix side inter mitochondrial membrane side what is the takehome message These proof of principle molecular motors 5 4539 demonstrated that the structurefunction relationships in 4 of 11 pages Bioc 460 Dr Miesfeld Fall 2008 the ATP synthase complex that catalyze ATP synthesis are the same ones that catalyze ATP hydrolysis figure 8 Figure 8 Intermem brane interrmembrane This brings us to the final mechanistic question how does proton movement through the 0 subunit ring of F0 cause rotation ofthe y subunit This is a good question however we need to rely more on a plausible explanation than the results of an elegant experiment to come up with a satisfactory answer at this point Figure 9 shows a proposed model for the Fo quotrotary enginequot based on ADPPi gt ATP ADppi ATP structural analysis ofthe yeast mitochondrial 0 subunit ring that was found to contain 10 identical subunits This quottwo channelquot model proposes that the a subunit contains a pair of proton channels each of which provide access to the 0 subunit ring from either the P positive side intermembrane space in a mitochondria or N negative side mitochondrial matrix sides ofthe membrane Importantly neither of the channels Figure 9 transverse the entire membrane and therefore function only as half channels Since the a Cytosolic halfchannel concentration of H on the P a side is higherthan it is on the N side a llp will readily enter the half channel in the a subunit where it then comes in contact with a negatively charged aspartate residue D61 in the Subunit 5 b a nearby c subunit Since protonation neutralizes the charge on the aspartate residue the c subunit ring is able to rotate into the hydrophobic membrane in the clockwise direction viewed from the Fo side ofthe membrane With this 40 clockwise rotation a proton bonded to D61 of the neighboring 0 subunit gains access to the second half channel in the a subunit and exits the channel due to the low HN concentration in the matrix This model for the Fo rotor is similar to a carousel ride at a carnival in which the carousel is the 0 subunit ring and the entrance and exit lines for the carousel are the two different proton channels in the a subunit As illustrated in figure 5 once a H enters from the P side it must ride the 0 subunit carousel once around until it is able to exit on the other side 1 Aspartic acid Matrix halfchannel Transport Systems in the Mitochondria A key element ofthe Chemiosmotic Theory is that the inner mitochondrial membrane must be impermeable to ions in order to establish the proton gradient Therefore biomolecules required forthe electron transport system and oxidative phosphorylation must be transported or quotshuttledquot back and forth across the inner mitochondrial membrane by specialized proteins The most important ofthese biomolecules are ATP ADP and Pi which are products and reactants in the ATP synthase reaction and cytosolic NADH a product of the gyceraldehyde3phosphate 5 of 11 pages dehydrogenase reaction in glycolysis Since most ATP consuming reactions take place in the cytoplasm but the majority of ATP synthesized by the cell occurs in the mitochondrial matrix the impermeability of the inner mitochondrial membrane creates a problem for the ATP synthase reaction Not only does newly synthesized ATP need to be exported from the matrix to replenish cytosolic ATP pools but the substrates for the reaction ADP and Pi need to be continually imported into the matrix to maintain high rates of ATP synthesis This is accomplished by two translocase proteins located in the inner mitochondrial membrane One is the ATPADP translocase also called the adenine nucleotide translocase which functions to export one ATP for every ADP that is imported and the other is the phosphate translocase which translocates one Pi and one H into the matrix by an electroneutral import mechanism figure 10 The ATPADP translocase is called an antiporter because it translocates molecules in opposite directions across the membrane for every Bioc 460 Dr Miesfeld Fall 2008 Figure 10 Intelmembrane Matrix space E5 9 quot7 i Adenine 1 7 nucleotich Ni V 4 A Li39auslt imse ADIll rantiporteri L ADI 9 l 7 g i ll e 39v I i If 7 ATP ll Tquot i 1 synthase 7 L F g l39 r z HEPCM PhOSDIIHTC HILigt39gK74Y translorzisv u Isymporterr H ll ADP molecule that is imported from the cytosol an ATP molecule is exported from the matrix The phosphate translocase is thought to function like a channel When the negatively charged Pi ion H2PO439 accompanies the positively charged H across the inner mitochondrial membrane in response to the proton gradient it is acting as a symporter because both molecules are translocated in the same direction This is an electroneutral translocation since the two charges H2PO439 and H cancel each other out Cytosolic NADH transfers electrons to the matrix via shuttle systems Numerous dehydrogenase reactions in the cytosol generate NADH one of which is the glycolytic enzyme glyceraldehyde F39g 11 3phosphate I we dehydrogenase However cytosolic NADH cannot cross the inner mitochondrial membrane Asparme instead the cell uses an indirect mechanism that only transfers the electron pair 2 e or two reducing equivalents from the cytosol to the matrix using two different quotshuttlequot systems The most widely used shuttle is the malateaspartate shuttle that has been found to operate in liver kidney and heart cells figure 11 Glutamate Aspartate A Glutamate transporter Glutamate gt7 Aspartate Cytosolic side uKetoglutarate Glutamate u Ketoglularate Glutamate Matrix side lt NADH H NAD39 Oxaloacetale 44 Malate x Ketoglutarate amm x Ketoglutarate transporter x Ketogluta rate Oxaloacetate 7 T Malate NADH H NAD39 Electron Transport System Bloc 460 7 Dr Miesfeid Fall 2008 The key enzymes in this shuttle pathway are cytosolic malate dehydrogenase which reduces oxaloacetate to malate and mitochondrial malate dehydrogenase the citrate cycle enzyme that oxidizes malate to form oxaloacetate In this way the electron pair picked up from the cytoplasmic NADH is transferred via malate which is transported across the membrane to a mitochondrial NAD molecule to form NADH that can then be oxidized by complex I in the electron transport system NADH reducing equivalents transported to the mitochondrial matrix by the malate aspartate shuttle are used to generate 25 ATP as a result of10 H being pumped across the inner mitochondrial membrane The malateaspartate shuttle is a bit more complicated than simply reoxidizing malate in the mitochondria to form oxaloacetate because sooner or later cytosolic oxaloacetate levels will be insuf cient to keep the shuttle going Therefore cytosolic oxaloacetate needs to be replenished which is accomplished by two additional enzymes mitochondrial aspartate aminotransferase and cytosolic aspartate aminotransferase as well as a Figure 12 second transporter that exchanges aspartate hiywlym for glutamate l The primary NADH shuttle in brain and muscle cells is the glycerol3 mm NADH w phosphate shuttle which differs from the malateaspartate shuttle in that the electron pair extracted from cytosolic NADH enters the electron transport chain at the point on WW 1 nion rather than complex I The result ofthis is llhnsplmlr that cytosolic NADH using this shuttle 1120 system can only produce 15 ATPNADH t llloll rather than 25 ATP because ofthe loss of4 H that are normally pumped across the membrane by complex I As shown in gure g the glycerol3phosphate shuttle consists of two isozymes of glycerol3phosphate dehydrogenase that function together to transfer 2 e from NADH in the cytosol to an enzymebound FAD molecule in the inner mitochondrial membrane The net yield of ATP from glucose oxidation in liver and muscle cells We have now completed our long journey through the central portion ofthe metabolic forest specifying aerobic energy conversion reactions that convert glucose to 002 H20 and of course ATP Let39s add everything up to see how one mole of glucose can be used to generate 32 ATP in liver cells via the malateaspartate shuttle or 30 ATP in muscle cells which use the glycerol3 phosphate shuttle As shown in gure 13 glycolysis produces 2 net ATP and 2 NADH while we get another 2 NADH when pyruvate is converted to acetyl CoA inside the mitochondrial matrix The citrate cycle generates 6 NADH 2 FADH and 2 GTP ATP from 2 acetyl CoA molecules bringing the total ofmitochondrial NADH to eight with another 2 NADH from the cytoplasm Oxidation ofthese 10 NADH by the electron transport system gives rise to 25 ATP in liver cells but only 23 ATP in muscle cells because the 2 cytosolic NADH in muscle cells donated their electrons to the FAD moiety of glycerol3phosphate dehydrogenase as described above We can now add in the 3 ATP derived from oxidation of the FADH contained in succinate dehydrogenase complex II the 2 ATP from glycolysis and the 2 ATP from the citrate cycle to give a grand total of 32 ATP in liver cells and 30 ATP in muscle cells Note that these net ATP yields 7 of M pages Bioc 460 Dr Miesfeld Fall 2008 from glucose oxidation Figure 13 should not be quot considered exact ATP Yield from Complete Oxidation of Glucose Value bePause they Process Direct product Final ATP are primarily based on GlyCOlySTS 2 CytOSUllCl Of 5 exchange ratios of 25 4 ill v ATPNADH and 15 F yruvate OXidation two per 2 NADH ll l l lOChO dFial 5 ATPFADH2 derived Wm mm from emplrlcal AcetyvaoA omclalion i NADH mitochondrial 15 determlnatlons 0f Citric aCId cyCie matrix proton flow through the two per glucose various components of 2 ITADH 3 the electron transport 2 Al P or 2 GTP 2 system and ATP Total yield bar glucose 30 or 22 synthase complex Tirie number depends on which shuttle sysier transfers reducing equivalents into mitochondria ETS and OxPhos are FunctionallyLinked As we saw in lecture 27 proton Figure 14 pumping by the ETS provides the proton motive force required for ATP synthesis Electron Transport figure 14 The functional System l1 linkage between the ETS and OxPhos pathways can be demonstrated by experiments using isolated mitochondria that are suspended in buffer containing 02 but lacking ADP Intermembrane space l 7quot i FADHZ 3 7 Furimi39ntr Pi and an oxidizable S quotquot L 39 substrate such as succinate W xixir Am 39 N which donates a pair of Matrix ox quot F1 i electrons to FAD in complex H 39H ATPsyn thaSe of the electron transport complex system Since maintenance of the proton gradient is required for the proton motive force that drives mm I conformational changes in the ATP Rotenone Antim Cm N3 synthase complex dissipation of the Amytal Myxothllazo CN DNP Aurovertin Atractyloside proton gradient leads to a decrease in Ptericidin Stigmatellin CO FCCP DCCD 3 k 9kicadd the rate of ATP synthesis Likewise blocking proton flow through the ATP synthase complex results in a proton gradient that is so large that rates substrate oxidation by the ETS are also decreased Figure 15 summarizes a 5 XidafiVe variety of synthetic and naturally Ph 5ph y quot quot ATPsynthase occurring inhibitors that have been 8 of 11 pages Bioc 460 Dr Miesfeld Fall 2008 shown to disrupt oxidative phosphorylation by either directly blocking the activity of the F1Fo ATP synthase complex oligomycin aurovertin venturicidin dicyclohexylcarboiimide DCCD or by inhibiting the electron transport system rotenone amytal piericidin antimycin myxothiazol stigmatellin cyanide CN39 carbon monoxide CO azide N3 Two other types of inhibitors that indirectly shutdown oxidative phosphorylation are the chemical uncouplers dinitrophenol DNP cyanidep trifluoromethoxyphenylhydrazone FCCP and inhibitors of the ATPADP translocase atractyloside bongkrekic acid which shut off the supply of ADP required for ongoing ATP synthesis As shown in figure 16 when ADP Pi are added to a mitochondrial suspension lacking succinate 02 reduction as measured by 02 consumption and ATP synthesis increase only slightly over time However when succinate is added then both the rates of 02 reduction and ATP synthesis increase dramatically until substrates become limiting Both 02 consumption and ATP synthesis are blocked when cyanide CN39 is added to the suspension since proton pumping by the electron transport system stops resulting in a shut down of the ATP synthase complex Similarly as shown in figure 17 addition of oligomycin which blocks proton flow through the ATP synthase complex not only inhibits ATP synthesis as would be expected but also leads to a decrease in 02 reduction since the energy required to pump protons across the membrane against the electrochemical proton gradient is greater than the energy released from the combined couped redox reactions of the electron transport system lmportantly addition of Figure 16 Add CN Add succinate Add ADP P 02 consumed ATP synllwsizm l Time Figure 17 Add oligomycin Add ADP Pi Add succinate Oz consumed Tl39 ltllll1l 5lzl ll Figure 18 H HP P HP HP dinitrophenol DNP to the suspension activates 02 reduction by lowering the energy barrier to proton pumping and thereby produces heat instead of A TP As shown in figure 18 DNP is a hydrophobic molecule that can easily diffuse across the membrane and in the process carry protons one a time from the intermitochondrial space high Hp to the matrix low H N In this regard DNP is functioning as a uncoupling agent because it uncoupes redox energy available from the electron transport system 02 consumption from ATP DinitrophenolDNP 07 l Hp Hp omit 11 7039 omit p H 7 lntermitochondrial membrane space Inner mitochondrial membrane Mitochondriamatrix pH8 N07 ozNie HTN HN HN 70 h OZN HI Diffusion N07 9 of11 pages EiDEAEUnDY Mtesteid Faiiznna synthests Uncoupiers such as DNP have been used as W diet piHS because they sttmuiate the body to oxidize tat tn 13 Nquot39 39 39 7 quot quot response to a chrontc state ot tow energy charge As you mtght tmagtne thts ts yety dangerous because DNP accumuiates tn the mttochondrtai membrane and can iead to sertous stde enects tnciudtng death Not at tnhtbttors ot oxidative phosphoryiatton are detrimentat tn tact eyoiutton has made good use ot the UCP1 uncoupling protein aiso caiied thermogenin to controithermogenests tn ammais CeHrspecific expression ot the UCPT protetn ieads to heat productton under aerobtc condtttons by short ctrcutttng the proton gradtent across the mttochondrtai tnner membrane as shown tn ttgure 19 The UCPT protetn ts expTeSSed at htgh ieyeis tn spectai tat ceiis caiied brown adipose tissue whtch contatn tatty actds torthe productton ot acetyi CoA to drtye NADH productton by the cttrate cycie and iarge numbers ot mttochondrta to tncrease the output ot heat by the eiectron transport system The brown coior ts these spectai tat ceiis ts due to cytochromes tn mttochondrtai protetns whtch ts the same reason stow twtch musctes are the dark meat ot a turkey tit ngcmm litm ANSWERS To KEY CONCEPT QUESTIONS IN OXIDATIVE BmSPHoRYLArIoN How does proton motive force drive ATP synthesis Proton flow through the Pya ATP synthase compex drtye ATP synthests oy tnductng contormattonat changes tn the Pya ATP synthase compex that decrease the amntty otthe enzyme for ATP tncreaaed AD The HR ATP synthase comptex can be broken down tnto three fundtnNaunttsthatworktogemerto harness the energy ot protonrmotive torce tor the purpose ot ATP synthests tthe rotor contatntngthe csubumt rtng andthey subunttwhtch turn togethertn a counteractockwiae dtrectton tooking at P1 trom the matrix stde as protons enter and exit the c subuntt rtng 2 the cataiyttc head piece contatntng the p subuntts and 3 the stator conststtng ot the a subuntt tmbedded tn the membrane and a stabtitztng arm made up ot the o dtmer and o subuntts whtch preyentsthe cataiyttc head ptece trom turntng when the c subuntt rtng and y subuntt rotate The btndtng change mechantsm modei proposes that rotatton ot the y subuntt tnduces three different contormattons tn the cataiyttc p subuntts caiied ioose Lt ttght T and open 0 whtch controi the btndtng ot ADP P to the L conformation the tormatton ot ATP tn theT conformation and the reiease ot ATP tn the o contormatton Accordtng to thts modet wtth every 3 H that pass through the FD subunit the y subuntt rotates mo and the p subuntts cycie through thethree contormattons L 77gt T 77gt o 77gt L productng 3 ATP tor eyety compiete 360 turn otthe rotor mom pages Bloc 460 Dr Miesfeld Fall 2008 Why does glucose oxidation in muscle cells produce two less ATP than in liver cells Complete glucose oxidation in muscle cells produces two fewerATP than liver cells because of the difference in NADH shuttle systems used in these two tissue types Glycolysis produces 2 NADH molecules in the cytosol for every glucose that is metabolized Liver cells utilize the malate aspartate shuttle which results in the net transfer ofthe electron pair from cytosolic NADH to mitochondrial NADH through the combined activity of cytosolic malate dehydrogenase and mitochondrial malate dehydrogenase which interconverts malate and oxaloacetate In contrast muscle cells utilize the glycerol3phosphate shuttle which transfers an electron pair from cytosolic NADH to FAD through a coupled reaction mechanism involving two isoforms ofthe enzyme glycerol3phosphate dehydrogenase Since oxidation of the FADH2 molecule in glycerol3 phosphate dehydrogenase by coenzyme Q bypasses complex I in the electron transport system the two cytosolic NADH molecules produced by glycolysis in muscle cells yield 2 less ATP molecules 30 ATP instead of 32 ATP than liver cells Explain why oligomycin inhibits succinate oxidation in isolated mitochondria Oligomycin blocks proton movement through the ATP synthase complex thereby increasing the electrochemical proton gradient and raising the energy barrier for continued proton pumping and succinate oxidation Oligomycin blocks the path for protons to ow down the electrochemical proton gradient back into the mitochondrial matrix Even though this does not directly block electron transfer through the ETS it results in a significant buildup of protons in the inter member space Since continued electron transfer from succinate to 02 is required to maintain high redox rates a large proton gradient will make it energetically unfavorable for redox reactions to occur and succinate will remain in the reduced state This can be demonstrated by adding dinitrophenol DNP to the mitochondrial suspension which will decrease the electrochemical proton gradient without coupled ATP synthesis and permit succinate oxidation to resume In contrast adding DNP to a mitochondrial suspension that has been treated with an ETS inhibitor such as cyanide has no effect on rates of succinate oxidation since electron transfer is blocked Note that addition of DNP or ETS or ATP synthase inhibitors all lead to a decrease in rates of ATP synthesis 110f11 pages
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