Class Note for BIOC 460 at UA 4
Class Note for BIOC 460 at UA 4
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Date Created: 02/06/15
Biochemistry 460 Dr Tischler OXIDATIVE PHOSPHORYLATION Related Reading Chapter 18 520524 527534 in Stryer 639h edition OBJECTIVES l Distinguish between electroneutral and electrogenic transport 2 Describe the signi cance of electrogenic transport for the adenine nucleotide transporter and the role of this transporter 3 List the features ofthe chemiosmotic model 4 Name the components of the ATP synthase complex and describe their roles 5 Discuss how the malate aspartate and the a glycerol phosphate differ Do not memorize the layout ofthe shuttle to reproduce it but understand their key aspects 6 De ne respiratory control and uncoupling and describe the physiological importance of these processes PHYSIOLOGICAL PREMISE Why do some snake and spider venoms cause cell death at the site of the bite in particular Some of these venoms contain enzymes called phospholipases Phospholipases hydrolyze membrane phospholipids to release fatty acids Phospholipases in snake or spider venom degrade phospholipids in the mitochondrial membrane The fatty acids released act as natural uncouplers that as is described in this lecture prevent oxidative phosphorylation by destroying the pH gradient Consequently the cell dies because of an inability to produce enough energy MITOCHONDRIAL TRANSPORT Electraneutral Transport Because the mitochondrial inner membrane is highly impermeable it contains many transport proteins to control the movement of substances into and out of the matrix Most substances move down their concentration gradient Thus when citrate is produced in large amounts in the matrix for fatty acid or cholesterol synthesis citrate generates a large concentration difference which favors its movement to the cytoplasm where it can participate in these pathways Most of the transport systems require the exchange of molecules and most of these exchanges occur with molecules having the same charge this is termed electroneutral transport For instance pyruvate 1 charge which enters the matrix for further metabolism via pyruvate dehydrogenase or pyruvate carboxylase exchanges with a negatively charged hydroxyl ion OH39 Phosphate PO439 which enters the matrix for synthesis of ATP see Fig 3 also can exchange with OH39 Citrate a tricarboxylic acid has a negative 3 339 charge at physiological pH but exchanges with malate a dicarboxylic acid 239 To maintain electroneutrality of this exchange a proton accompanies the citrate thus neutralizing one of its negative charges citrate339 If swaps with malatez39 Electragenic Transport There are some transport systems in which the exchange of molecules involves unequal charges When there is the net movement of charge across the membrane this is termed electrogenic transport In mitochondria net negative charges move out of the matrix while net positive charges must move into the matrix by following the charge gradient Recall that the pumping of protons creates a gradient of negative inside to positive outside so that the more negatively charged matrix will favor the net outward movement of negative charge Oxidative Phosphorylation l INNER Matrix Interrnembrane Space MEBNE slde I Electrogenic Translocases ATP L Adenine nucleotide translocase ADP339 1 Aspanatel39 Aspartate translocase Glutamate 39 H Figure 1 Electrogenic transport system in mitochondria The adenine nucleotide exchange ensures that ATP 39 produced in the mitochondrial matrix is transported to the cytoplasm where it is needed The exchange if aspartate in for glutamate out is unique because both have a negative one charge however a proton accompanies the glutamate to effectively neutralize its charge creating electrogenic exchange The most important electrogenic transporter is the adenine nucleotide transporter ATP produced in the mitochondrial matrix by oxidative phosphorylation see Fig 2 is needed in the cytoplasm for energy requiring processes such as muscle contraction lipogenesis cholesterol synthesis and gluconeogenesis Hence it is obligatory that ATP only be transported into the cytoplasm while ADP moves into the mitochondria matrix where it can be phosphorylated to ATP ADP bears a negative three charge whereas ATP has a negative four charge at physiological pH Fig 1 Thus the exchange of ATP 39 moving out with ADP339 moving in creates a net charge of negative one moving into the interrnembrane space The charge gradient facilitates this exchange of ADP for ATP The reverse exchange cannot occur in functional mitochondria because it would require the more negative ATP molecule to move towards the more negative environment of the matrix a process that cannot occur Similarly the glutamateaspartate translocase is electrogenic because glutamatel outside is neutralized by cotransport of a PP into the matrix in exchange for aspartatel moving out This translocase is an integral part of the malateaspartate shuttle described later in this lecture Electrogenic translocases are irreversible as long as a charge gradient exists across the membrane more negative inside because the protons pumped out are positive Electrogenic transport requires energy but ensures unidirectional outward ux of ATP and aspartate under normal circumstances OXIDATIVE PHOSPHORYLATION THE CHEMIOSMOTIC MODEL Overview Oxidative phosphorylation consists of two processes The oxidative portion is the respiratory chain and is recreated in the right hand side of g 2 Shown is the movement of protons through complexes 1 III and IV creating a proton and charge gradient across the inner mitochondrial membrane This gradient is then used to provide energy to produce ATP the phosphorylation part of the process lower left of Fig 2 described below The charge gradient facilitates the inward transport of ADP and the ef ux of ATP upper left of Fig 2 described later in this lecture Oxidative Phosphorylation 2 Chemiasmatic Model Pr ADP3 INTERMEMBRANE SPA CE 4H NADH FMNH2 quotquotme39ex39 H OH39 e inner 3H NAD membrane P39 ADP339 A ATP L G i I COQ complex Iquot H e cytb 4 2H lZO C1 MATRIX c aa3 C H20 4H gt Proton gradient Charge gradient 3H lt ATP SYcheSiS transporters O respiratory chain components components Figure 2 Asymmetrical orientation of electron transport chain components in inner membrane Heavy dark lines trace electron e ow For complex I the reduced aVin mononucleotide prosthetic group FMNHZ is indicated Complex II II donates electrons to coenzyme Q CoQ but does not pump protons like complexes 1 III and IV The heme prosthetic groups of complex 111 b and c1 are indicated Cytochrome C the link between complexes III and IV is attached to the outer surface intermembrane side of the inner mitochondrial membrane Oxidative Phosphorylation 3 The chemiosmotic model explains the requirements and features of oxidative phosphorylation as follows 1 The mitochondrial inner membrane system is impermeable to protons IF by simple diffusion 2 A measurable proton gradient exists across the inner membrane 3 Collapse of the proton gradient uncoupling abolishes ATP synthesis but accelerates oxygen consumption discussed below 4 Inhibition of the respiratory chain prevents ATP synthesis because pumping of protons ceases ATP synthuse complex see orientation in inner membrane Fig 2 F1 ATPase located on the inside of the inner membrane extends into the matrix It contains the catalytic sites for ATP synthesis Inward proton ow use of energy from the pH proton gradient allows the ATPase to synthesize ATP F0 is the integral membrane protein attached to F1 via the stalk The F0 contains the channel that opens to allow protons to ow down their gradient into the matrix The stalk regulates proton ow and ATP synthesis Oligomycin is an antibiotic that inhibits ATP synthesis by binding to F0 The most recent data suggest that energy derived from the inward ow of 3 protons is required for the synthesis of one molecule of ATP However the export of this ATP molecule from the matrix requires 1 proton so that the true cost of ATP formation and movement to the cytoplasm maybe 4 protons Consequently the oxidation of each molecule of NADH that pumps out 10 protons can in theory generate just 25 ATP Oxidation of FADHZ which leads to the pumping of just 6 protons could only generate 15 ATP The traditional estimate of energy production has been 3 ATP per NADH and 2 ATP per FADHZ molecule For our purposes the most important aspect of this discussion is the comparison of NADH versus FADHZ and aerobic versus anaerobic oxidation of glucose No matter whether one uses the traditional estimate or the more quantitative approach two facts still remain First is that oxidation of NADH generates an additional molecule of ATP compared to FADHZ Second is that the complete oxidation of glucose produces considerably more ATP than can be generated by anaerobic glycolysis ELECTRON SHUTTLE SYSTEMS Under aerobic conditions NADH produced during glycolysis must be oxidized by the mitochondria In this way the cell regenerates NAD for the glyceraldehyde3phosphate dehydrogenase reaction and produces additional energy by feeding electrons to the respiratory chain Since there is no transporter to move NADH directly into the matrix oxidation of cytoplasmic NADH by mitochondria must occur indirectly either via the malate aspartate Fig3 or the oc glycerol phosphate Fig4 electron shuttle Malate uspartate shut e Figure 3 NADH from glycolysis produced in muscle heart brain and other tissues with high energy demands is oxidized by the malateaspartate shuttle Electrons from cytoplasmic NADH are used to reduce oxaloacetate to malate which is then transported into the matrix Thus malate carries the electrons from NADH into the mitochondria In the matrix NADH is produced by the oxidation of malate to oxaloacetate in the citric acid cycle via malate dehydrogenase This shuttle is irreversible because the transport of aspartate out of the matrix in exchange with glutamate moving into the matrix is electrogenic just as is the exchange of ATP with ADP While these amino acids normally carry the same charge the exchange is electrogenic because a proton neutralizes to zero the negative charge on the glutamate Thus aspartatel39 exchanges for glutamate0 so that there is a net outward movement of a negative charge It is important that this transporter operate in only one direction unidirectional to ensure that electrons from mitochondrial NADH do not move to the cytoplasm in these tissues Oxidative Phosphorylation 4 e39 eectrons Glucose INNER OUTER MEMBRANE e liAD GLYCOLYSIS MEMBRANE e A I Pyruvate I OAA NADH N AD Complex I 0AA Glue gt Glu e39 1 5 e 6 Asp Asp1 4 l 1 3 I e39 e39 NAD Malate Malate NAD CYTOPLASM MATRIX OAA Oxaloacetate Glu glutamate Asp aspartate KG ocketoglutarate Figure 3 The malateaspartate shuttle Cytoplasmic malate dehydrogenase 1 reduces oxaloacetate OAA to malate The ocketoglutarate KG transporter 2 exchanges malate for KG Mitochondrial malate dehydrogenase 3 generates intramitochondrial NADH by oxidation of malate to oxaloacetate Mitochondrial aspartate aminotransferase 4 catalyzes the transfer of an amino group from glutamate glu to oxaloacetate to produce KG and aspartate asp KG is transported out on its translocase 2 and aspartate is transported out on the unidirectional aspartate translocase 5 Cytoplasmic aspartate aminotransferase 6 regenerates oxaloacetate for reaction 1 by transferring the amino group from aspartate to KG producing glutamate which is transported into the matrix in exchange for aspartate 5 Glycerol Phosphate Shut e Figure 4 The glycerol phosphate shuttle occurs almost exclusively in the liver Electrons from cytoplasmic NADH reduce dihydroxyacetone phosphate to glycerol phosphate which in turn carries the electrons to the respiratory chain Electrons reach the respiratory chain via glycerol phosphate dehydrogenase that is bound to the inner membrane This enzyme contains a FAD prosthetic group as we showed for succinic dehydrogenase of the citric acid cycle Thus electrons are fed directly to coenzyme Q Since complex I is bypassed this shuttle produces one fewer ATP from glycolytic NADH than occurs with the malate aspartate shuttle Also unlike the other shuttle the electron carrier glycerol 3phosphate never permeates the inner membrane but interacts instead with the transmembrane glycerol 3phosphate dehydrogenase Oxidative Phosphorylation 5 OUTER INNER MEMBRANE MEMBRANE e eectrons 2 Glucose N AD GLYCOLYSIS MATRIX Pyruvate Dihydroxyacetone NAD phosphate DHAP e39 DHAP Glycerol 1 3 phosphate Glycerol G3P dehydrogenase NAD 3phosphate CYTOPLAS M Figure 4 Glycerol phosphate shuttle Cytoplasmic glycerol 3phosphate dehydrogenase 1 oxidizes NADH Glycerol 3phosphate dehydrogenase in the inner mitochondrial membrane 2 reduces bound FAD to FADHZ CONTROL OF MITOCHONDRIAL RESPIRATION OXYGEN CONSUMPTION Respiratory Control The rate of respiration is ultimately controlled by the availability of ADP in the mitochondrial matrix This is referred to as respiratory control ADP binding to the F1 signals the stalk to open the proton channel Since inorganic phosphate is abundant the concentration of ADP determines when ATP is to be synthesized When energy demands are high the concentration of ADP will be high in this cell The elevated concentration of ADP in the mitochondrial matrix will promote the inward movement of protons for ATP synthesis As protons are transferred inward down their gradient to support ATP synthesis the respiratory chain responds by pumping out more protons in conjunction with an increased rate of respiration O2 consumption In this way ADP provides control of respiration When the concentration of ADP is low such as occurs at rest respiration slows because the pH gradient reaches a maximum amount and more protons cannot be pumped out In the presence of inhibitors of the respiratory chain respiratory control is lost because protons can no longer be pumped out even when the concentration of ADP 1ncreases Oxidative Phosphorylation 6 Uncaupling Figure 5 GLYCOGEN GLUCOSE LACTATE CYTOPLASM NADHA NADH ALANINE A PYRUVATE 39 MALATE ASP SHUTTLE INTERMEMBRANE I SPACE P C PY UVATE I PDH ACE YL CoA MATRIX complex HI AT I FF I ADP P1 complex I g I uncoupler K ATP ADP Pi Figure 5 The effects of uncoupling on glucose lactate and mitochondrial metabolism Uncouplers are substances eg natural uncoupling proteins fatty acids or chemicals such as dinitrophenol DNP that bind protons and are hydrophobic Because of their hydrophobicity these substances can diffuse across the inner mitochondrial membrane despite its relative impermeability By carrying protons into the matrix uncouplers destroy collapse or dissipate the pH gradient by equilibrating the concentration of protons across the membrane This causes ATP formation to cease because there is no longer an existing pH gradient to drive ATP synthesis However oxygen consumption remains rapid because the respiratory chain continues to attempt to maintain a pH gradient albeit unsuccessfully Remember that the rate of the respiratory chain actually responds to the size of the pH gradient the smaller the gradient the faster the rate Thus arti cial dissipation of the gradient creates a constant rapid rate of respiration The glycolytic pathway will operate rapidly in conjunction with the malateaspartate shuttle quickly oxidizing any NADH that forms Pyruvate will be preferentially oxidized to CO2 rather than reduced to lactate because NADH amounts are low Oxidative Phosphorylation 7 Since the energy from the respiratory chain redox reactions cannot be coupled to ATP synthesis instead it is released as heat and the body temperature rises Rapid O2 consumption in uncoupling represents a loss of respiration control With uncoupling and no ATP synthesis 0 use is very rapid as the mitochondrion futilely attempts to generate a proton gradient As noted in the physiological premise fatty acids can act as uncouplers Certain venoms contain phospholipases that release fatty acids causing uncoupling of mitochondria in that cell and potentially necrosis of the tissue tissue death that contains cells attacked by the venom While uncoupling can have pathological consequences there are natural uncoupling processes that are important for nonshivering heat production This is particularly important in infants who generally have underdeveloped shivering re exes Located near major organs eg liver heart is a concentration of brown adipose tissue so named because of its high abundance of mitochondria This tissue contains special uncoupling proteins that as the name implies uncouple mitochondria Since brown adipose tissue mitochondria are relatively uncoupled they produce heat rather than ATP and thus help maintain the temperature of critical organs As we grow the total amount of brown adipose tissue remains constant and therefore eventually represents an insigni cant mass of tissue in the body The use of brown fat becomes unnecessary because the shivering re eX and other heat generating mechanisms become operative Summa of uncoupling effects 0 rate of respiration rises because the pH gradient is arti cially collapsed by the uncoupler o collapse of the pH gradient prevents ATP synthesis from occurring 0 rapid rate of respiration reduces the amount of mitochondrial NADH relieving inhibition of the citric acid cycle and of pyruvate dehydrogenase o mitochondrial ADP increases because of ATP hydrolysis 0 increased ADP activates isocitrate dehydrogenase and pyruvate dehydrogenase o decline in mitochondrial NADH leads to decreased cytoplasmic NADH so that lactate formation from glucose decreases Medical Scenario 1 As a current medical student and a former lawyer you become interested in what could have been a lucrative malpractice case in the 1920 s At that time some physicians without the bene t of modern biochemistry knowledge prescribed dinitrophenol as a weightreducing agent For some patients it worked satisfactorily the only side effects being sweating and a slight elevation in temperature However in patients who were more overweight and required longterm treatments their body temperature increased to such an eXtent that death resulted Associated with their condition was a marked increase in their rate of respiration and an inability to gain weight even with increased caloric intake Medical Scenario 11 During World War 11 workers in munitions factories were eXposed to a chemical called trinitrophenol A common occurrence in these workers was frequent absenteeism due to elevated temperature weakness and accelerated breathing that improved after several days at home What would you have recommended to reduce the incidence of such illness OXidative Phosphorylation 8
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