Class Note for BIOC 460 at UA
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Date Created: 02/06/15
Oxidative Phosphorylation Lecture 30 Key Concepts Structure and function ofthe ATP synthase complex Transport systems in mitochondria Regulation of oxidative phosphorylation How does proton flow through the F1F0 ATP synthase complex drive ATP synthesis Why does glucose oxidation in muscle cells produce two less ATP than in liver cells Biochemical Application of the Oxidative Phosphorylation The F1 component of the ATP synthase complex can be used as a quotnanomotorquot to drive ATP synthesis by attaching a magnetic bead to the gamma subunit and forcing clockwise rotation using electromagnets Nano biotechnology is a rapidly growing eld that utilizes biomolecules to build molecular machines Magnelic bead Slreplawdin any complex HS lag The ATP Synthase Comples uses the proton motive force generated via the electron transport system to synthesize ATP through conformational changes in a process called oxidative phosphorylation ATP Amino Acid Metabolism Carbohydrate Metabolism l Glycolysis Lipid Metabolism A Cavbon Fixation Photosynthesis V EIEKI DH J Transport ATP Currency Exchange Ratios of NADH and FADH2 Experimental measurements demonstrate 3 H are required to synthesize 1 ATP when they flow back down the electrochemical proton gradient through the ATP synthase complex and 1 H is needed to transport each negativelycharged Pi molecule into the matrix 1 NADHaxidized 10 H quotoutquot ATP 4b ADP 1 ATP synthesized 4 H quotinquot ATPADP lranslocase P 1 W 39 Phosphate lranslocase kl 39 NADH H ADP i 1 H10 K ATP K i 3 29 NAD P 1 W 31202 f Eifcran 7 4 4 ATPsynihase Transport complex System 10H 3H ATP Currency Exchange Ratios of NADH and FADH2 Taking into account the requirement of 3 HATP synthesized and the use of 1 H to translocate ADP We can now see where the ATP currency exchange ratios of 25 ATPNADH and 15 ATPFADH2 come from oxidation of NADH by complex I leads to 10 HI4 H 25 ATP oxidation of FADH2 by complex yields 6 WM H 15 ATP for FADHZ Structure and Function of the ATP Synthase Complex ATP synthase complex represents one ofthe quintessential protein machines found in living cells Mitochondrial ATP synthase complex consists of two large structural components called F1 which encodes the catalytic activity and F0 which functions as the proton channel crossing the inner mitochondrial membrane Three functional units of ATP Synthase the rotor rotates as protons enter and exit the ring the catalytic head piece contains the enzyme active site in each ofthe three beta subunits the stator consists of the a subunit imbedded in the membrane which Stator k contains two half channels for protons to enter and exit the F0 component and a stabilizing arm 3D view movie A 3D view of alpha3beta3gamma Click here to go to UC Berkeley site for ATP synthase movies Proton flow through FO alters the conformation of F1 subunits The realization that the catalytic activity of the three beta subunits was regulated by conformational changes induced by the rotating g subunit provided the key to understanding the enzyme mechanism of the F1F0 ATP synthase complex Nucleotide binding studies revealed that it was the affinity of the beta 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 presence of proton motive force the dissociation constant KD decreased by a millionfold 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 B subunits control ATP production Top view movie A top view of alpha3beta3gamma Click here to go to UC Berkeley site for ATP synthase movies 1 The binding change mechanism incorporates three basic principles The gamma subunit directly contacts all three beta subunits however each of these interactions are distinct giving rise to three different B subunit conformations The ATP binding af nities of the three beta 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 B subunit is in the O conformation As protons flow through F0 the gamma subunit rotates in a counter clockwise circle looking at F1 from the matrix side such that with each 120 rotation the b subunits sequentially undergo a conformational change from L gt Tgt O gt L The binding change mechanism model predicts that one full rotation of the gamma subunit should generate 3 ATP since each beta subunit will have cycled once through the T state 120 rotation of y counterclockwise gt Cross section view movie A crosssection view of alphabetagamma Click here to go to UC Berkeley site for ATP synthase movies Note that the ratio of 3 HIATP is not yet certain because there are unanswered questions regarding the molecular mechanism ofthe 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 see lecture 29 both of which have been empirically determined Boyer39s model predicts that ATP hydrolysis by the F1 headpiece should reverse the direction of the v subunit rotor To test this idea Masamitsu Yoshida and Kasuhiko Kinosita of Tokyo Institute of Technology used recombinant DNA methods to modify the alpha beta and gamma subunits of the E coli F1 component in order to build a synthetic molecular motor Movie atpmov How does proton movement through the c subunit ring cause rotation of the y subunit A proposed model for the F0 quotrotary enginequot is shown below based on structural analysis ofthe east mitochondrial c subunit rin that was found to contain 10 identical su units Since the concentration 0 H on the P side positive side intermembrane space is higher than it is on the N side negative side matrix a HP 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 nearby c subunit H Cytosolic halfchannel Aspartic acid Matrix halfchannel Subunit c Subunit a A Carousel Ride at a Carnival the carousel is the c subunit ring the entrance and exit lines for the carousel are the two different proton channels in the a subunit H H w H39 H H H H H H H H H H H r H H H H H H H H H H H H39 H39 w H H H H H H Intermembrane H H space V Can rotate H IOEkWiSE Matrix Cannot rotate Ht in either direction Transport Systems In The Mitochondria Key element of the Chemiosmotic Theory The inner mitochondrial membrane must be impermeable to ions in orderto establish the proton gradient Biomolecules required for the electron transport system and oxidative phosphorylation must be transported or quotshuttledquot back and forth across the inner mitochondrial membrane by specialized proteins Accomplished by two translocase proteins located in the inner mitochondrial membrane Two Translocase Proteins 1 ATPADP Translocase also called the adenine nucleotide translocase functions to export one ATP for every ADP that is imported an antipon er because it translocates molecules in opposite directions across the membrane for every ADP molecule that is imported from the cytosol an ATP molecule is exported from the matrix 2 Phosphate Translocase translocates one Pi and one H into the matrix by an electroneutral import mechanism ATPADP Translocase Cytosolic side i l k k A l ADPr ADP Evelsiun 9 gt ADP 7 Matti side 7 ADPm ATP ATP ATPquot Eversion ATP Phosphate Translocase Thought to function like a Intergl 39zg39me 5 quotkmquot channel A When the negatively V E Ad charged PI Ion HzPo 39 US329 A1 pquot ltquotquot my accompanies the positively u snslumse Ami 39 r 7 charged H across the inner anlipm39lerl re 1 Ami mitochondrial membrane in it sg response to the proton gradient it is acting as a ATP H symponer because both Sh39ll mse molecules are translocated in the same direction This is an electroneutral r le translocation sincethe two rhusf mu H2P01 739 mmlucusn V charges HZPO and H lsympn ery H H cancel each other out is Cytosolic NADH transfers electrons to the matrix via shuttle systems 0 Numerous dehydrogenase reactions in the cytosol generate NADH one ofwhich is the glycolytic enzyme glyceraldehyde3phosphate dehydrogenase 0 However cytosolic NADH cannot cross the inner mitochondrial membrane 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 Most widely used shuttle is the malateaspartate shuttle Found to operate in liver kidney and heart cells and functions as a reversible pathway The key enzymes in this shuttle pathway are cytosolic malate dehydrogenase that reduces oxaloacetate to malate and mitochondrial malate dehydrogenase the citrate cycle enzyme that oxidizes malate to form oxaloacetate W Cylosolk side 4 NADH 7 H NAD39 Aspanm W Oxaloacelate LA Malala xeroglmaraze Glulamale wkemglutavate Glutamate A gramme Grummaxe mummy Malaler mmmglummrp r rransparrer uAKemglularate Glutamate urKemglutavate Glutamate Asparme A Dxaloacetate 7T Mame Malrlxside NADH H NM 3 Flertmn Transport swam The primary NADH shuttle in brain and muscle cells is the glycerol3 phosphate shuttle Glyculysis Differs from the malate aspartate shuttle the electron pair extracted from va mm in cytosolic NADH enters the electron transport chain at 39 I h Lulu3 h is the point of Q rather than 5119011 complex I V L mymm rr thydruxyncelmua LH 0 The result Of this IS that whowham nhosnh dw 3 cytosolic NADH using this H20H shuttle system can only tmm produce 15 ATPNADH rather than 25 ATP because of the loss of 4 H that are normally pumped across the membrane by complex I Matrix 1 l The net yield of ATP from glucose oxidation in liver and muscle cells Let39s add everything up to see how one mole of glucose can be used to generate 32 ATP in liver cells via the malate aspartate shuttle or 30 ATP in muscle cells which use the glycerol3 phosphate shuttle ATP Yield from Complete Oxidation of Glucose Process Direct product Final ATP Glycolysrs 2 NADH cytosolic 3 or 5 2 ATP 2 Pyruvate cxrdation two per 2 NADH mitochondrial 5 glucose matrixl AcetylCoA oxidation in 6 NADH mitochondrial 15 Citric acid cycle matrix two per glucose 2 FADHg 3 2 ATP or 2 GTF 2 Total yield per glucose 30 or 32 39The number depends on which shultle system transfers reducing equivalents into mitochondria Regulation of Oxidative Phosphorylation Cellular concentration of ADP is a key control factor of oxidative phosphorylation Determines the rate of ATP synthesis the rate of NADH oxidation and reduction of O2 to form H20 The regulatory function of ADP and ATP in controlling aerobic respiration extends to the citrate cycle and glycolysis both of which are activated by a low energy charge high ADP and low ATP Regulation of Oxidative Phosphorylation The ratio of NADHNAD in the mitochondrial matrix controls multiple steps in the citrate cycle This in turn determines the ow of electrons through the electron transport system and ultimately rates of ATP synthesis Regulation of Oxidative Phosphorylation The role of the electrochemical proton gradient in linking substrate oxidation to ATP synthesis can be demonstrated by experiments using isolated mitochondria that are suspended in buffer containing 02 but lacking ADP Pi and also lacking an oxidizable substrate such as succinate which would donate a pair of electrons to FAD in complex ll of the electron transport system When ADP Pi are added 02 consumption and ATP synthesis increase only slightly over time When succinate is added both the rates of O2 consumption and ATP synthesis increase dramatically until substrates become limiting Both 02 consumption and ATP synthesis are blocked when cyanide CN is added to the suspension since proton pumping bythe electron transport system stops resulting in a shut down of the ATP synthase complex Regulation of Oxidative Phosphorylation Add CN Add succinate Add ADP Pi 02 consumed ATP synthesized Time Isolated mitochondria in buffer containing 02 but lacking ADP Pi and also lacking an oxidizable substrate such as succinate Add DNP Add oligomycin Add ADP P 02 consumed Add snccinate ATP synthusized Time Two other inhibitors are oligomycin which inhibits the ATP synthase reaction by blocking proton flow through the proton channel and DNP dinitrophenol a hydrophobic molecule that diffuses across the membrane and in the process dissipates the proton gradient by carrying protons one a time from the inter mitochondrial space high H to the matrix low H OH 0 DNP functions as a uncoupling A NOQ rl No agent because it uncouples redox 4 f H energy available from the electron 1 transport system 02 consumption N01 NOZ from ATP SyntheSiS Z39llinitmulieiml DNP The UCP1 uncoupling protein Inlermemhrnnc Manix 5pm also called thermogenln controls thermogenesis in animals Cellspecific expression of the UCP1 protein leads to heat production under aerobic conditions by short circuiting the proton gradient across the mitochondrial inner membrane The UCP1 protein is expressed at high levels in special fat cells called brown adipose tissue which contain fatty acids for the production of acetyl CoA to drive NADH production by the citrate cycle and large numbers of mitochondria to increase the output of heat by the electron transport system How is this different from the Alternative l fr t g jln x Oxidase in Plants ithermogcnim Hem
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