Class Note for BIOC 460 at UA 6
Class Note for BIOC 460 at UA 6
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Date Created: 02/06/15
Bioc 460 Dr Miesfeld Spring 2008 Electron Transport System Supplemental Reading Key Concepts PETER MITCHELL 39S CHEMIOSMOTIC THEORY THE ELECTRON TRANSPORT SYSTEM IS A SERIES OF COUPLED REDOX REACTIONS Complex I NADHubiquinone oxidoreductase Complex II Succinate dehydrogenase Complex lll Ubiquinonecytochrome c oxidoreductase Cytochrome C Complex IV Cytochrome c oxidase BASIS FOR THE A TP CURRENCY EXCHANGE RATIOS OF NADH AND FADH2 KEY CONCEPT QUESTIONS IN THE ELECTRON TRANSPORT SYSTEM What is the Chemiosmotic Theory and how does it explain proton motive force What is the role of coenzyme Q ubiquinone in the electron transport system Biochemical Application of the Electron Transport Svstem 0 Hydrogen cyanide is a deadly gas that kills cells by blocking 0 electron transfer from cytochrome oxidase in complex IV to oxygen Other electron transport inhibitors are rotenone a poison and amytal a barbiturate both of which block electron transfer A mgfggfoggg gg i ghe from ironsulfur centers e39mntmspomvmm The Electron Transport System also called the Electron Transport Chain converts redox energy available from oxidation of NADH and FADH2 into protonmotive force which is used to synthesize ATP through conformational changes in the ATP synthase complex through a process called oxidative phosphorylation The relationship of the electron transport system and oxidative phosphorylation to other metabolic pathways in illustrated in figure 1 Figure 1 Carbohydrate PETER MITCHELL39S CHEMIOSMOTIC THEORY Mam Oxidation of NADH and FADH2 in the mitochondrial matrix by the electron transport system links redox energy to ATP synthesis by 1 Magggm oxidative phosphorylation mitochondrial ATP synthesis through the establishment of a proton H gradient across the mitochondrial WW3 i j inner membrane This quotchemiosmoticquot process was first proposed by avggggoggg N i Peter Mitchell a British biochemist and involves the outward pumping of H from the mitochondrial matrix by three protein complexes in the electron transport system complexes I III IV The protons flow back down the gradient through the membrane bound ATP synthase complex in response to a chemical H concentration and electrical separation of charge differential This Tg zm um same chemisomotic process generates the bulk of ATP used by bacterial cells which are thought to be the ancestors of eukaryotic ATP mitochondria and is used to synthesize ATP in plant chloroplasts which utilize sunlight energy rather than redox energy to establish a H gradient across the thylakoid membrane The electron transport system consists of the linked redox reactions that occur sequentially in a set of protein complexes imbedded in the inner mitochondrial membrane The net result of sz NADH Carbon Fixation Electron Transport Photosynthesis J 02 H20 1 of 10 pages Bioc 460 Dr Miesfeld Spring 2008 these redox reactions is the coupled oxidation of NADH and reduction of molecular oxygen 02 to form NAD and H20 The difference in reduction potential AE 39 between the NADHNAD conjugate redox pair E 39 032V and that of the 02H20 conjugate redox pair E 39 082V provides a huge amount of free energy for ATP synthesis 220 kJmol Figure 2 summarizes the electron transport system and the process of chemiosmosis as it applies to mitochondria Figure 2 ll iii A ll l Xvi vi lnternwmbrnnc A gt gt I v 39l quot 39 space i M w l l 39 i i In 3 l l i H u 77 39li iii TIL J w 7 quotgt7 a iim 39 39 39l r llmmmh m 11 7 I hun39limbr lllJ l1 39 NAM ii i I lquot quotr H A Mnmx xquot Iquot m A l39i v quot 39 il Ulwmlwl A39l39l Elwmcul pntvllliul synLIuma pmumiui ApH tlrnvLnhy All iinshln protnnmntivv llnsilll39 alkaline l nm npgmivel The basic idea of the chemiosmotic theory now generally accepted as being correct is that energy from redox reactions or light is translated into vectoriaenergy through the coupling of electron transfer to membrane bound proton pumps that transverse a proton impermeable membrane and thereby establish an Figure 3 electrochemical proton gradient A quotproton ProtonC rcuit circuitquot is established when the protons H respond to the chemical and electrical AP gradient across the membrane by flowing H my back across the membrane through the ATP synthase protein complex to catalyze ATP axPhos Matrix 02 55 synthesis as shown in figure 3 e J H7O Note that the vectorial H pumping results in ADP 1 AW39t P both a chemical gradient across the membrane represented by ApH and an Memb ane electrical gradient due to the separation of charge which can be measured as a membrane potential A14 The separation of charge is due to the buildup of positivelycharged protons HT on one side ofthe membrane and accumulation of negative charges on the other side ofthe membrane OH39 Note that in mitochondria the contribution of Au to AG is actually greater than that of ApH the ApH across the mitochondrial membrane is only 1 pH unit whereas in chloroplasts the ApH contribution to AG is much more significant with ApH close to 3 pH units 2 of 10 pages Bioc 460 Dr Miesfeld Spring 2008 Figure 4 As shown in gure 4 a critical feature of the mitochondrion is the extensive surface area of the inner mitochondrial membrane which forms the proton impermeable barrier required for chemiosmosis This highly convoluted membrane is the location of electron transport system proteins and the ATP synthase complex and therefore maximum surface area is required to accommodate as much protein as possible Electron microscopy studies have shown that the inner mitochondrial membrane forms structures called cristae which have been estimated to cover as much as 3000 if per cell 5 uz per mitochondrion Cristae Intermembrane space Matrix Outer membrane Inner membrane Mitchell39s chemiosmotic theory was eventually validated using biochemical approaches This was done using quotinsideoutquot membrane vesicles that could Figure 5 be shown to pump protons Into the Interior of the Bacteriorhodopsin in vesicle when oxidizable substrate was made available Synthetic veside H leading to the production of ATP at the surface of the vesicle exactly as predicted by Mitchell39s theory As illustrated in gure 5 one type of experiment used synthetic membrane vesicles consisting of a 41 mixture of phosphatidylethanolamine and phosphatidylcholine that were reconstituted in the presence of puri ed bacteriorhodopsin protein from the photosynthetic bacteria Halobacterium halobium and bovine heart mitochondrial membrane fractions consisting of the intact ATP synthase complex When the vesicles were exposed to light proton Mitochondrial pumping by the bacteriorhodopsin protein resulted in ATPase both inward proton pumping and ATP synthesis on the vesicle surface The bacteriorhodopsin proton pump is analogous to the electron transport system except proton pumping is initiated by light rather than oxidation of NADH and FADH2 CADP P AjP We are now ready to start ourjourney through the metabolic forest by answering our four key questions about the quotpathwayquot that links the electron transport system to oxidative phosphorylation 1 What does the electron transport systemoxidative phosphorylation accomplish for the cell Generates ATP derived from oxidation of metabolic fuels accounting for 28 out of 32 ATP 88 obtained from glucose catabolism Tissuespecific expression of uncoupling protein1 UCP1 in brown adipose tissue of mammals shortcircuits the electron transport system and thereby produces heat for thermoregulation 2 What is the overall net reaction of NADH oxidation by the coupled electron transport and oxidative phosphorylation pathway 2 NADH 2 H5ADP 5 Pi029 2 NAD5ATP 2 H20 3 of 10 pages Bioc 460 Dr Miesfeld Spring 2008 3 What are the key enzymes in the electron transport and oxidative phosphorylation pathway ATP synthase complex the enzyme responsible for converting protonmotive force energy available from the electrochemical proton gradient into net ATP synthesis through a series of protondriven conformational changes NADH dehydroenase also called complex I or NADHubiquinone oxidoreductase This enzyme catalyzes the rst redox reaction in the electron transport system in which NADH oxidation is coupled to FMN reduction and pumps 4 H into the intermembrane space Ubiquinonecytochrome c oxidoreductase also called complex III translocates 4 H across the membrane via the Q cycle and has the important role of facilitating electron transfer from a two electron carrier QH2 to cytochrome c a mobile protein carrier that transfers one electron at a time to complex IV Cytochrome c oxidase also called complex IV pumps 2 H into the inter membrane space and catalyzes the last redox reaction in the electron transport system in which cytochrome a3 oxidation is coupled to the reduction of molecular oxygen to form water 02 2 e 2 H gt H20 San Quentin as chamber 4 What is an example of the electron transport system Cyanide binds to the heme group in cytochrome a3 of complex IV and blocks the electron transport system by preventing the reduction of oxygen to form H20 Hydrogen cyanide gas is the lethal compound produced in prison gas chambers when sodium cyanide crystals are dropped into sulfuric acid THE ELECTRON TRANSPORT SYSTEM IS A SERIES OF COUPLED REDOX REACTIONS Early work using puri ed mitochondria showed by fractionation methods and biochemical assays that ve enzyme complexes contained within the inner mitochondrial membrane were required for oxidative phosphorylation These components were named Complex I NADHubiquinone oxidoreductase NADH dehydrogenase Complex II succinate dehydrogenase Complex Ill Ubiquinonecytochrome c oxidoreductase and Complex IV cytochrome c oxidase The fth enzyme fraction contained the F1Fo ATP synthase complex actually puri ed as an ATP hydrolyzing activity consisting of a large multisubunit complex which could be further fractionated into a membrane bound quotstalkquot F0 and a spherical quotheadquot F1 encoding the catalytic subunit Using specific redox reaction inhibitors such as rotenone antimycin A and cyanide and based on the known reduction potentials E 39 of conjugate redox pairs it was possible to order the four electron transport system complexes as shown in figure 6 Figure 6 Inlvrmvmhmnv Cytochrome 111 mm mm Q Cytochrome c oxidase 4quot NADH oxidoreductase 4quot 7 1 dehydrogenase 94 T 39quot 39yquot T v I l 9 z r 7IA2H39 no NAUHHT Mir ShlL LlliiltL Fm me Matrix 5 sum Succi nate dehydrogenase 4 of 10 pages atoc bBEIrDl Mtestelo SPllnEZDDB Metapolte toel tn the torm ot NADH and FADH teeo tnto the Elemrun transport systemtromthe ettrate cycle and tatty aeto oxtoatton pathways Falls ot Elemruns 2 er are donated by NADH and FADHz to eomplexl and H lespectlvely ano tlowthroogh the electron transport system onttl they are used to reduce uxygerl to torm water co2 2 e 2 H 77gt H20 The Wu mobile electron carriers tn thts sertes ot reaettons are coenzyme Q m also called ubiquinone and cytochrome c Mlch transter eleetrons between yanoos complexes The stotehtometry ot pluturl pumping ts 4 H tn eomplex L4 Hlrl umplexlll and 2 H tn eomplex lv lEI H lNADH and a H39FADH2 The oonatton ot 2 e trom NADH tn the torm ot a hyonoe ton H39 to FMN tntttates a sertes ot seooenttal reoox reaettons tnyolytng as many aszo discrete electron carriers eolmtnattng tn the reooetton ot moleeolar uxygen to torm Water Falls ot Elemruns as hydrogen atoms 2H39 2e can also enter the Elemrun transport system throogh oxtoatton ot FADH2 moleeoles uvalentlyr attached to enzymes assoetateo vnth ettherthe eytosolte stoe otthe tnner mttoehononal memorane mttoehononal glycerulrarphusphate oehyorogenasey orthat pelong to metapolte pathways loeateo thhtn the mttoehononal matrtx soeetnate oehyorogenase ano ETFVQ uxlduleductase The four toncttonal components otthe electron transport system are Three large multisubunit protein complexes l Iquot and IV that transyerse the tnner mttoehononal memprane ano tonetton asproton quotpumasquot Coenzyme Q at also called ubiqulnuney a small hyorophopte Elemrun earnerthat dlffuses lateraly wtthtn the membrane to non te electrons to complex lll Three membranerassuclated FADcontai ing enzymes soeetnate oehyorogenaset Electrunr transferrlrlg avupruteln ETF ano glycerulrarphusphate oehyorogenase thatptch up electrons from ltnhed metabolc pathways and donate them to eoenzyme Q Cytochrome c a small wateresolople protetn that assoetateswtth the eytosolte stoe ot the membrane and carrtes electrons one at a ttmetrom eomplex lll to complex lv Flgure 7 beluvv shows the reooetton potenttals tor many ot the Elemrun earners tn thts system Standard Reductton Potentials nf Respiratory Chain and Related Electron Carriers Rudax median lhzllewacllnnl Equot W 2n 2s a H a clc NllD H t 2e NADH 0320 Nnnp t e 2e annuph oszc NADad Integerescltltht t 2H t e Mkl39m denydrrginas lr39hYNHl nototttne 2h tze gooootntt tyrotnrtmeolre t t e ty nthrtmibl e l t enromecttie e ew nrtntec Fa t Cyi chrtm le t a le romeotre l t e rylcchmmeelFe l Ly39mchm i a L attoenrtmectre t e ytocnromee ot2ll 2e 7 lie Huvv ts the energy released by reoox reaettons used to pump protons tnto the lrlterrmembrarle spaee7 The answer ts not yet completely understood pot tt tsthooght to tnyolyet l a redox loop mechanism tn whteh there ts a separatton otthe H and e on oppostte stoes otthe membrane 5 min paoes Bioc 460 Dr Miesfeld Spring 2008 the Q cycle in complex lll uses this mechanism to translocate protons across the membrane and 2 redoxdriven conformational changes in the protein complex that quotpumpquot protons across the membrane by altering pKa values of functional groups located on the inner and outer faces ofthe membrane Both complexes l and IV have properties that are consistent with such a proton pumping mechanism It is likely that for some electron transport proteins both a redox loop mechanism and protein conformational changes are involved in net proton movement across the membrane Figure 8 illustrates how these two mechanisms could be operating Figure 8 Redox Loop Mechanism 2 4 Proton Pump mechanism Complex Ill Q cycle Complexes l and IV Complex I NADHubiquinone oxidoreductase Complex is the largest ofthe four protein complexes in the mitochondrial electron transport system consisting of as many as 42 polypeptide chains having a combined molecular mass of 850 kDa Because of its complexity and size the structure of complex I is not yet known at the molecular level however electron microscopy methods have provided a low resolution map of its physical contour which looks a bit like a sideways quotLquot as shown in gure 9 The function of complex I is to pass 2 e obtained from the oxidation of NADH to Q using a coupled W 1 reaction mechanism that results in the net movement of 4 H across the membrane Complexl Intelmeqbrane Complex contains a covalently bound flavin 7 9393 s de mononucleotide FMN that accepts the two 39 11 electrons from NADH as well as at least six l f N 39 l different ironsulfur centers FeS that carry i 1 one electron at a time from one end ofthe complex to the other The poison rotenone blocks electron transfer within complex I by m 39 preventing a redox reaction between two FeS i centers Complex passesthe two electrons obtained from NADH as a hydride ion H39 to Q NADH NAD ubiquinone which has three critical roles in the electron transport system 1 Q serves as a mobile electron carrier that transports electrons laterally in the membrane from complex I to complex Ill 2 Q is the entry point into the electron transport system for electron pairs 2 e obtained from the citrate cycle fatty acid oxidation and the enzyme glycerol3P dehydrogenase and 3 Q has the important task of converting a 2 e transport system into a 1 e transport system which passes electrons one at time to the mobile Matrix N side 6 of 10 pages Bloc 460 7 Dr Mlesfeld Spring 2008 carrier protein cytochrome c This conversion process 2 e gt 1 e 1 e is accomplished by the Q cycle as described later Complex II Succinate dehydrogenase Succinate dehydrogenase is a citrate cycle enzyme Figure 10 we rst encountered in lecture 28 It catalyzes an mumm lecemi oxidation reaction that converts succinate to fumarate Srimosvhm in a coupled redox reaction involving FAD This mme A enzyme was copuri ed along with other polypeptides Vqx that together constitute complex ll of the electron 1725 transport system The 2 e extracted 39om succinate quotl A in the citrate cycle is passed through the other protein FMN t subunits In the complex to Q as shown In gure 10 J No protons are translocated across the inner Vm q m W mitochondrial membrane by complex The gt mm quot quot H electron pair donated by succinate to FAD ultimately Mm FMWMVH DA leads to the translocation of four fewer H than NADH because complex II is not a proton pump This is why the ATP currency exchange ratio for FADH oxidation is lower than it is for NADH giving rise to only 15 ATPFADH instead of 25 ATPNADH The same holds true for electron pairs extracted from the FADH moiety of glycerol3P dehydrogenase and ETFQ oxidoreductase both of which donate 2 e to Q Complex III ubiquinonecytochrome c oxidoreduciase Complex III is the docking site for QH2 ubiquinol and consists of 11 protein subunits in each of two monomer subunits Figure 11 shows a diagram of complex lll emphasizing the relative position ofthe electron carriers and the presence of two distinct binding sites for ubiquinone called GP and ON which play a crucial role in diverting one electron at a time to cytochrome c via the 0 cycle The terms GP and ON refer to the proximity of the sites to the positive intermembrane space and negative matrix sides of the membrane The molecular structure ofa complex lll monomer is also shown in gure 11 illustrating its orientation in the inner mitochondrial membrane M mummy quotlm llxumxl 7 or 10 pages Bioc 460 Dr Miesfeld Spring 2008 The four steps in the Q cycle are shown in figure 12 and can be summarized as 1 Oxidation of QHz at the Qp site results in transfer of one electron to the Rieske FeS center which is transferred to cytochrome C7 and then passed off to Cyt o The second electron is transferred to cytochrome bL which quotstoresquot it temporarily The oxidation of QH2 in this first step contributes 2 Hp to the intermembrane space 2 The oxidized Q molecule moves from the Qp site to the QN site through a proposed substrate channel within the protein Figure12 complex This stimulates electron 2H transfer from bL to bH which then 1 Translocate to reduces Q in the QN site to form the 7 7 A r Complexlv semiquinone Qquot intermediate Q 4 r is i 3 A new QHz molecule binds in the vacated Qp site and is oxidized in the same way as step 1 such that one electron is transferred to cytochrome C7 and then to a new molecule of Cyt o Oxidation of this second QH2 molecule translocates another 2 Hp into the inter membrane space 4 Hp total and the resulting Q molecule is released into the membrane the QN site is occupied with Qquot 4 The second electron from the QHz oxidation in step 3 is passed directly from bL to bH and then used to reduce the semiquinone Qquot intermediate already sitting in the QN site which uses 2 Hm to regenerate a QH2 molecule Translocate to Complex IV To see how the Q cycle accomplishes the 2 e gt 1 e 1 9 conversion process is to write outtwo separate QH2 oxidation reactions and then sum them to getthe net reaction for complex lll OH Cyt o oxidized gt Qquot 2 H p Cyt 0 reduced QH Qquot 2 HM Cvt c oxidized gt Q QHz 2 H p Cvt 0 reduced QHz 2 Hm 2 Cyt o oxidized gt Q 4 H p 2 Cyt 0 reduced Note that the Q cycle reactions require that 2H N from the matrix be used to regenerate QHZ even though 4Hp are translocated However this apparent imbalance of2H N is corrected by the redox reactions of complex N where 2H N are required to reduce oxygen to water and 2H N are pumped across the membrane Therefore the nettranslocation of protons across the membrane in the combined redox reactions of complexes Ill and IV becomes 6 Hm gt 6 Hp Cytochrome C Cytochrome c Cyt o is a small protein of 13 kDa that associates with the cytosolic side ofthe inner mitochondrial membrane and is responsible fortransporting an electron from complex III to complex lV using an ironcontaining heme prosthetic group Oxidized Cyt 0 contains ferric iron 8 of 10 pages Bioc 460 Dr Miesfeld Spring 2008 Fe3 in the heme group and reduced Cyt 0 contains ferrous iron Fe2 The heme group of Cyt c is covalently attached to the protein through cysteine residues and Figure 13 lies Within a pocket surrounded by three D helices The molecular structure ofa typical Cyt c protein is shown in gure 13 A version of the Cyt c molecular structure is used in the Bioc460 website header Complex IV Cytochrome c oxidase Complex IV accepts electrons one at a time from Cyt c and donates them to oxygen to form water In the process two net H are pumped across the membrane using a conformationaltype Hemegmp mechanism similarto complex I The mitochondrial complex IV protein consists of two monomers of 200 kDa that each 59 contain 13 polypeptides two copper centers CuA and Gus nimmmhmne and two heme groups cytochrome a and cytochrome a3 73251 739 As shown in gure 14 Cyt c docks on the P side ofthe membrane to complex IV near CuA which accepts the electron leading to oxidation ofthe heme group in Cyt c Fe2 gt Fe The reduced CuA passes the electron to an iron atom in the heme of cytochrome a which then transfers it to cytochrome a3 Finally the e is passed to CuB which donates it to oxygen BASIS FOR THE ATP CURRENCY EXCHANGE RATIOS OF NADH AND FADH2 Considering that 3 Hp are required to synthesize 1 ATP when they ow back down the electrochemical proton gradient through the ATP synthase complex and 1 Hp is 2 H 2 W H O M r needed to transport each negativelycharged Pi molecule Autismmi ipumpmli 2 trillx into the matrix see gure 15 we can now see where the ATP currency exchange ratios of 25 ATPNADH and 15 ATPFADH2 come from Namely oxidation of NADH by complex I leads to 10 Hp4 HN 25 ATP whereas oxidation of FADH2 by complex II yields 6 Hp4 HN 15 ATP for FADH2 Figure 15 1 NADH oxidized 10 H1L quotoutquot ATP H ADP 1 ATP synthesized 4 H in ATpADp translocase Pi 1H 7 Phosphate Iranslocase ADP H 20 NADH H ATP 5 57gt NAD P 1 H 1202 Electron ATPsanhase ag550 complex 8 1o H 3 H 9 of 10 pages Bloc 460 Dr Miesfeld Spring 2008 ANSWERS TO KEY CONCEPT QUESTIONS IN THE ELECTRON TRANSPORT SYSTEM The Chemiosmotic Theory was rst proposed by Peter Mitchell in 1961 and states that enerqy captured by coupled redox reactions in the electron transport system is used to translocate pump protons across the inner mitochondrial membrane creatinq a protonmotive force ApH my that drives ATP synthesis by proton ow throuqh the HE ATP synthase complex The H are pumped across the membrane by complex I 4 H complex Ill 4 H and complex IV 2 H The return of these 10 H to the mitochondrial matrix in response to protonmotive force results in the production of25 ATPNADH This ATP currency exchange ratio is based on the assumption that 3 H must pass through the F1Fo ATP synthase complex for every ATP generated and that 1 H is required to import Pi into the matrix through the phosphate translocase 10 Hl4 H 25 Coenzyme Q ubiquinone is a hydrophobic molecule that functions as a mobile electron carrier that transports a pair of electrons 2 e in its reduced form of QH2 ubiquinol from either complex I or from membranebound FADH2containinq enzymes such as succinate dehydroqenase or qucerol3phosphate dehydroqenase to complex I The oxidation of NADH by complex results in the extraction of 2 e that pass through a series of FeS centers in the complex until being donated to Q to form QH2 which then travels to complex Ill and donates one electron at a time to cytochrome c using via the Q cycle Oxidation of FADH2 by Q bypasses complex I and therefore results in only 6 H being pumped across the membrane in the reduction of Oz to form H20 4 H from complex Ill and 2 H from complex IV The difference of1 ATP in the ATP currency exchange ratio of NADH and FADH2 is the result ofdirect oxidation of Q downstream of complex I loss of4 H 1 ATP 10 of 10 pages
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