BCHEM 254 METABOLISM IN HEALTH AND DISEASES II Lecture 1 The Energetics of the Electron Transport Chain Christopher Larbie, PhD

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1 BCHEM 254 METABOLISM IN HEALTH AND DISEASES II Lecture 1 The Energetics of the Electron Transport Chain Christopher Larbie, PhD Introduction The Citric Acid cycle oxidizes acetate into two molecules of CO2 while capturing the electrons in the form of 3 NADH molecules and one molecule of FADH2. These reduced molecules contain a pair of electrons with a high transfer potential. These electrons are ultimately going to be transferred by a system of electron carriers to O2 to form H2O. This process occurs in the mitochondria and is the major energy source used to produce ATP by oxidative phosphorylation. Let s look at the standard free energy change for the overall reaction shown below: NADH + H + + ½O2 NAD + + H2O NAD + + 2e - + H + NADH E o = V ½O2+ 2e - + 2H + H2O E o = V ΔE o = V ( V) = V ΔG o = nfδe o = 219 kj/mol ADP + Pi ATP ΔG o = kj/mol If we could convert all of this free energy generated by reducing oxygen with NADH to synthesize ATP from ADP and Pi: #ATP = 219/30.5 = 7.2 ATP Mitochondria The processes involved in electron transport and oxidative phosphorylation occur within membranes. Prokaryotes do not have organelles such as mitochondria. Bacteria have a plasma membrane surrounded by a rigid cell wall. In prokaryote systems, electron transport and oxidative phosphorylation are carried out across the plasma membrane. 1 P a g e D r. C h r i s t o p h e r L a r b i e

2 Eukaryotes have organelles including mitochondria. Mammalian cells have 800 to 2,500 mitochondria per cell. Remember that red blood cells do not contain mitochondria. Mitochondria are remarkably mobile organelles. Time lapse photography shows that they are constantly moving and changing shape. In some cells they are anchored by attachment to the cell s cytoskeleton so that they remain fixed at one cellular location to target a site of high ATP utilization. In heart muscle for example the mitochondria are anchored close to the contractile muscle, in sperm they are wrapped tightly around the motile flagellum. Shown above is a mitochondrion. The mitochondrion is enclosed by an outer membrane and a more complex inner mitochondrial membrane. The space between the inner and outer mitochondrial membranes is called the intermembrane space. Within this space we find enzymes that utilize ATP such as creatine kinase and adenylate kinase. The outer membrane contains porins which are transmembrane proteins rich in β- sheets which allow molecules of low molecular weight (<10,000) freely diffuse in and out. As a result, the outer membrane is like a sieve that is permeable to all molecules less than 10,000 Daltons. This makes the intermembrane space chemically equivalent to the cytosol of the cell with respect to small molecules. The inner mitochondrial membrane is packed with proteins which account for 80% of the membranes molecular weight. The inner membrane is impermeable to molecules and ions. Metabolites that must cross the inner mitochondrial membrane are carried across by specific transport proteins. The inner membrane is folded into numerous cristae, which greatly increase the surface area of the inner membrane. Within this membrane are the proteins involved in the electron transport chain, ATP synthase and transport proteins. 2 P a g e D r. C h r i s t o p h e r L a r b i e

3 The large internal space enclosed by inner mitochondrial membrane is called the matrix. It is densely packed with hundreds of enzymes, including pyruvate dehydrogenase, pyruvate carboxylase, and the soluble enzymes of the citric acid cycle. In addition there are the enzymes involved in the oxidation of fatty and amino acids. The matrix also contains the mitochondrial genome, the mitochondrial ribosomes, trna s and the enzymes required for the expression of mitochondrial genes. The Components of the Electron Transport Chain The electron transport chain of the mitochondria is the means by which electrons are removed from the reduced carrier NADH and transferred to oxygen to yield H2O. 1. NADH NADH is generated in the matrix by the reactions of pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehyrogenase. The electron transport chain begins with reoxidizing NADH to form NAD + and channelling the electrons into the formation of reduced coenzymes. Important to note that NADH transfers 2 electrons at a time in the form of a hydride. NAD + + 2e- + H + NADH E o = V 2. Flavoproteins Flavoproteins have either a FAD (flavin adenosine dinucleotide) or a FMN (flavin mononucleotide) prosthetic group. Flavoproteins can accept or donate electrons one or two at a time. Thus they are often intermediaries between two electron acceptors/donors and one electron acceptors/donors. For flavoproteins the typical standard reduction potentials are around 0 V. FAD + 2e - + 2H + FADH2; FMN + 2e - + 2H + FMNH2 E o 0 V 3. Coenzyme Q (CoQ) (or ubiquinone (UQ)) CoQ has ten repeating isoprene units which make it insoluble in water, but soluble in the hydrophobic lipid bilayer. Coenzyme Q is a versatile cofactor because it is a soluble electron carrier in the hydrophobic bilipid layer of the inner mitochondrial membrane. Like flavoproteins, CoQ can accept/donate electrons one at a time or two at a time. Q + 2e - + 2H + QH2 Q + e - + H +.QH.QH + e - + H + QH2 E o =0.060 V E o =0.030 V E o =0.190 V 3 P a g e D r. C h r i s t o p h e r L a r b i e

4 4. Cytochromes Cytochromes are proteins that contain haem prosthetic groups which function as one electron carriers. The haem iron is involved in one electron transfers involving the Fe 2+ and Fe 3+ oxidation states. Cytochromes are named by their absorption spectra which depends on the porphyrin structure and environment. The example shown is the haem prosthetic group of cytochrome b. Cytochrome b contains the same iron porphyrin found in haemoglobin and myoglobin. Other cytochromes we will encounter in the electron transport complexes are cytochromes b, c, c1, a and a3. 5. Iron-Sulphur Proteins In the electron transport chain we will encounter many iron-sulphur proteins which participate in one electron transfers involving the Fe 2+ and Fe 3+ oxidation states. These are non-haem iron-sulphur proteins. The simplest iron-sulphur protein is Fe-S in which 4 P a g e D r. C h r i s t o p h e r L a r b i e

5 iron is tetrahedrally coordinated by four cysteine residues. The second form is Fe2S2 which contains two irons complexed to 2 cysteine residues and two inorganic sulphides. The third form is Fe3S4 which contains 3 iron atoms coordinated to three cysteine residues and 4 inorganic sulphides. The last form is the most complicated Fe4S4 which contains 4 iron atoms coordinated to 4 cysteine residues and 4 inorganic sulphides. 6. Copper Proteins Copper bound proteins participate in one electron transfers involving the Cu + and Cu 2+ oxidation states. Overview of the Electron Transport Chain Electrons move along the electron transport chain going from donor to acceptor until they reach oxygen, the ultimate electron acceptor. The standard reduction potentials of the electron carriers are between the NADH/NAD + couple ( V) and the oxygen/h2o couple (0.816 V). The components of the electron transport chain are organized into 4 complexes. Each complex contains several different electron carriers. 1. Complex I also known as the NADH-coenzyme Q reductase or NADH dehydrogenase. 2. Complex II also known as succinate-coenzyme Q reductase or succinate dehydrogenase. 3. Complex III also known as coenzyme Q-cytochrome c reductase or coenzyme Q reductase. 4. Complex IV also known as cytochrome c reductase. Complex I accepts electrons from NADH and serves as the link between glycolysis, the citric acid cycle, fatty acid oxidation and the electron transport chain. Complex II includes succinate dehydrogenase and serves as a direct link between the citric acid cycle and the electron transport chain. Complexes I and II both produce reduced coenzyme Q, CoQH2 which is the substrate for Complex III. There are other two ways 5 P a g e D r. C h r i s t o p h e r L a r b i e

6 to feed electrons to UQ: the electron-transferring flavoprotein, which transfers electrons from the flavoprotein-linked step of fatty acyl-coa dehydrogenase, and snglycerophosphate dehydrogenase. Complex III transfers the electrons from CoQH2 to reduce cytochrome c which is the substrate for Complex IV. Complex IV transfers the electrons from cytochrome c to reduce molecular oxygen into water. Each of these complexes are large multisubunit complexes embedded in the inner mitochondrial membrane. 6 P a g e D r. C h r i s t o p h e r L a r b i e

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8 Complex I Complex I is also called NADH-Coenzyme Q reductase because this large protein complex transfers 2 electrons from NADH to coenzyme Q. Complex I was formerly known as NADH dehydrogenase. Complex I is huge, 850,000 kd and is composed of more than thirty subunits. It contains a FMN prosthetic group and seven or more Fe-S clusters. This complex has between iron atoms bound. The prosthetic group FMN is absolutely required for activity. Therefore this complex is a flavoprotein. This complex binds NADH, transfers two electrons in the form of a hydride to FMN to produce NAD + and FMNH2. The subsequent steps involve the transfer of electrons one at a time to a series of iron-sulphur complexes that includes both 2Fe-2S and 4Fe-4S clusters. Note the importance of FMN. First it functions as a 2 electron acceptor in the hydride transfer from NADH. Second, it functions as a 1 electron donor to the series of iron sulphur clusters. FMN and FAD often play crucial links between 2 electron transfer agents and 1 electron transfer agents. 8 P a g e D r. C h r i s t o p h e r L a r b i e

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10 The final step of this complex is the transfer of 2 electrons one at a time to coenzyme Q. CoQ like FMN and FAD can function as a 2 electron donor/acceptor and as a 1 electron donor/acceptor. CoQ is a mobile electron carrier because its isoprenoid tail makes it highly hydrophobic and lipophillic. It diffuses freely in the bilipid layer of the inner mitochondrial membrane. The process of transferring electrons from NADH to CoQ by complex I results in the net transport of protons from the matrix side of the inner mitochondrial membrane to the inter membrane space where the H + ions accumulate generating a proton motive force. The intermembrane space side of the inner membrane is referred to as the P face (P standing for positive). The matrix side of the inner membrane is referred to as the N face. The transport of electrons from NADH to CoQ is coupled to the transport of protons across the membrane. This is an example of active transport. The stiochiometry is 4 H + transported per 2 electrons. Complex II Believe it or not, you are already familiar with Complex II. It is none other than succinate dehydrogenase. The only enzyme of the citric acid cycle that is an integral membrane protein. This complex is composed of four subunits. 2 of which are ironsulphur proteins and the other two subunits together bind FAD through a covalent link to a histidine residue. These two subunits are called flavoprotein 2 or FP2. Complex II contains 3 Fe-S centres, 1 4Fe-4S cluster, 1 3Fe-4S cluster and 1 2Fe-2S cluster. In the first step of this complex, succinate is bound and a hydride is transferred to FAD to generate FADH2 and fumarate. FADH2 then transfers its electrons one at a time to the Fe-S centres. Thus once again FAD functions as 2 electron acceptor and a 1 electron donor. The final step of this complex is the transfer of 2 electrons one at a time to coenzyme Q to produce CoQH2. The overall reaction for this complex is: Succinate Fumarate + 2H + + 2e - CoQ + 2 H + + 2e - CoQH2 10 P a g e D r. C h r i s t o p h e r L a r b i e

11 Net Succinate + CoQ Fumarate + CoQH2 ΔE o = V (+0.031V) = V ΔG o = nfδe o = 5.6 kj/mol Compare this with complex I NADH + H + + CoQ NAD + + CoQH2 ΔE o = V ( 0.315V) = V ΔG o = nfδe o = 72.4 kj/mol For complex II the standard free energy change of the overall reaction is too small to drive the transport of protons across the inner mitochondrial membrane. This accounts for the 1.5 ATP s generated per FADH2 compared with the 2.5 ATP s generated per NADH. Other flavoproteins can also supply electrons to UQ, including mitochondrial sn-glycerophosphate dehydrogenase, an inner membrane-bound shuttle enzyme, and the fatty acyl-coa dehydrogenase, three soluble matrix enzymes involved in fatty acid oxidations. Complex III This complex is also known as coenzyme Q-cytochrome c reductase because it passes the electrons from CoQH2 to cyt c through a very unique electron transport pathway called the Q-cycle. Cytochrome b contains the same iron protoporphyrin as haemoglobin and myoglobin. The c cytochromes contain haem c through covalent attachment by cysteine residues. Cytochrome a is found in two forms in complex IV. In complex III we find two b-type cytochromes and one c-type cytochrome. Complex III is complex and we have a crystal structure. Complex III has a beautiful dimeric structure. The bottom of the structure extends 75 Å into the mitochondrial matrix, while the top of the structure extends 38 Å out into the intermembrane space. Shown in pale green are the α helices of cytochrome b which define the transmembrane portion of the complex. Shown in bright yellow is the Reiske protein which is an iron-sulphur protein that is mobile in the crystal structure. This motility is required for the electron transfer function of this protein. 11 P a g e D r. C h r i s t o p h e r L a r b i e

12 Q-Cycle The Q-cycle is initiated when CoQH2 diffuses through the bilipid layer to the CoQH2 binding site which is near the intermembrane face. This CoQH2 binding site is called the QP site. The electron transfer occurs in two steps. First one electron from CoQH2 is transferred to the Rieske protein (a Fe-S protein) which transfers the electron to cytochrome c1. This process releases 2 protons to the intermembrane space. Coenzyme Q is now in a semiquinone anionic state, CoQH. - still bound to the QP site. 12 P a g e D r. C h r i s t o p h e r L a r b i e

13 The second electron is transferred to the cyt bl haem which converts CoQH. - to CoQ. This reoxidized CoQ can now diffuse away from the QP binding site. The bl haem is near the P-face. The bl haem transfers its electron to the bh haem which is near the N- face. This electron is then transferred to second molecule of CoQ bound at a second CoQ binding site which is near the N-face and is called the Qn binding site. This electron transfer generates a CoQ. - radical which remains firmly bound to the Qn binding site. This completes the first half of the Q cycle. 13 P a g e D r. C h r i s t o p h e r L a r b i e

14 The second half of the Q-cycle is similar to the first half. A second molecule of CoQH2 binds to the QP site. In the next step, one electron from CoQH2 (bound at QP) is transferred to the Rieske protein which transfers it to cytochrome c1. This process releases another 2 protons to the intermembrane space. The second electron is transferred to the bl haem to generate a second molecule of reoxidized CoQ. The bl haem transfers its electron to the bh haem. This electron is then transferred to the CoQ. - radical still firmly bound to the Qn binding site. The take up of two protons from the N-face produces CoQH2 which diffuses from the Qn binding site. This completes Q cycle. The net result of the Q-cycle is 2e - transported to cytochrome c1. Two protons were picked up from the N-face in the second half of the Q-cycle and 4 protons total were released into the intermembrane space. The two electron carrier CoQH2 gives up its electrons one at a time to the Rieske protein, the bl and bh haem both of which are 1 electron carriers. The electrons that end up on cytochrome c1 are transferred to cytochrome c. Cytochrome c is the only water soluble cytochrome. Cytochrome c is coordinated to ligands, histidine nitrogen and to the sulphur of methionine residues that protect the iron contained in the haem from oxygen and other oxidizing agents. Cytochrome c is a mobile electron carrier that diffuses through the intermembrane space shuttling electrons from the c1 haem of Complex III to CuA site of complex IV. 14 P a g e D r. C h r i s t o p h e r L a r b i e

15 Cytochrome c shown above. The haem is linked to the protein by 4 cysteine linkages shown in yellow. A methionine sulphur atom is coordinated to the iron complexed in the haem. A histidine residue protects the iron from oxygen and other potential ligands. Complex IV Cytochrome c oxidase Complex IV is also known as cytochrome c oxidase because it accepts the electrons from cytochrome c and directs them towards the four-electron reduction of O2 to form 2 molecules of H2O. 4 cyt c (Fe 2+ ) + 4 H + + O2 4 cyt c (Fe 3+ ) + 2H2O 15 P a g e D r. C h r i s t o p h e r L a r b i e

16 Cytochrome c oxidase contains 2 haem centres, cytochrome a and cytochrome a3 and two copper proteins. Each of the protein bound coppers are associated with one of the cytochromes. The copper sites are called CuA and CuB. CuA is associated with cytochrome a and is shown to the left. CuB is associated with cytochrome a3. The copper sites function as 1 electron carriers cycling between the cuprous state Cu + and the cupric state Cu 2+ just like iron containing proteins they transfer electrons one at a time. Cytochrome c is bound on the P-face of the membrane and transfers its electron to CuA. The oxidized cytochrome c dissociates. CuA then transfers the electron to cytochrome a. The protein bound CuA and the iron bound in cytochrome a are 15 Å apart. In contrast the CuB, the iron bound in cytochrome a3 are very close to each other forming a binuclear metal centres shown below. Cyt a transfers the electron to CuB. A second cytochrome c binds and transfers its electron to CuA which is subsequently transferred to cytochrome a which in turn is transferred to cytochrome a3. The binuclear metal centres now has two electrons bound allowing the binding of O2 to binuclear centres. The next step involves the uptake of two protons and the transfer of yet another electron through the same pathway which leads to cleavage of the O-O bond and the generation of a Fe 4+ metal centres. The fourth electron is transferred to form a hydroxide at the haem centres which becomes protonated and dissociates as H2O. The mechanism is shown below. 16 P a g e D r. C h r i s t o p h e r L a r b i e

17 The reduction of oxygen by complex IV involves the transfer of four electrons. Four protons are abstracted from the matrix and two protons are released into the intermembrane space. Chemiosmotic Coupling The four major complexes of the electron transport chain operate independently in the inner mitochondrial membrane. Each of the complexes is an aggregate of proteins that are held firmly together by noncovalent forces. There is no experimental evidence that the four complexes associate with one another in the membrane. Each of these complexes has its own rate of lateral diffusion through the bilipid membrane which shows that the complexes do not move together. Kinetic studies of reconstituted electron transport systems support the theory of four independent complexes. However, evidence supports the existence of multimeric supercomplexes or respirasomes. Mutation studies suggest that Complex III is required to maintain Complex I in the mitochondria of human and mouse cells, and Complex IV is necessary for the proper assembly of Complex I in mouse fibroblasts. Further evidence suggests the requirement of cardiolipin in the assembly and stability of supercomplexes. 17 P a g e D r. C h r i s t o p h e r L a r b i e

18 The model for the electron transport system is shown above. Four independent complexes are transferring electrons through the mobile electron carriers of CoQ and cytochrome c. CoQH2 is produced by both complex I and complex II and delivers the electron to complex III via the Q-cycle. Complex III reduces cytochrome c which is a water soluble electron carrier located in the intermembrane space of the mitochondria. The reduced cytochrome c carries the electrons to complex IV which transfers the electrons to molecular oxygen. The process of electron transfer is coupled to transporting protons from the matrix to the intermembrane space of the mitochondria. 18 P a g e D r. C h r i s t o p h e r L a r b i e

19 This generates a chemical potential and an electrostatic potential. This potential energy is used to drive the synthesis of ATP. This is known as the Mitchell s Chemiosmotic hypothesis. Complex I: NADH + 5H + N + Q NAD + + QH2 + 4H + P Complex II: FADH2 + Q FAD + QH2 Complex III: QH2 + 2H + N + 2 Cyt c (Fe 3+ ) 2 Cyt c (Fe 2+ ) + Q + 4H + P Complex IV: 4Cyt c (Fe 2+ ) + 8 H + N + O2 4 Cyt c (Fe 3+ ) + 4H + P + 2H2O Overall reaction beginning with NADH: NADH + 11H + N + ½O2 NAD H + P + H2O (10H + P/2e - ) Overall reaction beginning with FADH2: FADH2 + 6H + N + ½O2 FAD + 6H + P + H2O (6H + P/2e - ) NADH + H + + ½O2 NAD + + H2O NAD + + 2e - + H + NADH E o = V ½O2+ 2e - + 2H + H2O E o = V ΔE o = V ( V) = V ΔG o = nfδe o = 219 kj/mol FADH2 + ½O2 FAD + H2O FAD + 2e - + H + FADH2 E o V ½O2+ 2e - + 2H + H2O E o = V ΔE o = V (0 V) = V ΔG o = nfδe o = kj/mol Most of the free energy from the transfer of electrons from NADH or FADH2 to molecular oxygen has been used to pump electrons out of the matrix across the inner mitochondrial membrane into the intermembrane space. Recall that the electrochemical energy inherent is this difference in proton concentration. ΔG = RTln(C2/C1) + ZFΔψ where C2/C1 is the concentration ratio for the ion being transported, Z is the absolute value of the ions electrical charge which is 1 for a proton and Δψ is the transmembrane electrical potential measured in volts. When one talks about hydrogen ion concentrations, one usually talks in terms of ph = log[h + ]. The log function (base 10) and the natural log function (base e) are related by the following relationship. Ln(x) = 2.303log(x) ΔG = RTlog([H + P] /[H + N]) + ZFΔψ ΔG = 2.303RT (log [H + P] - log[h + N]) + ZFΔψ ΔG = RT(-pHP + phn) + ZFΔψ 19 P a g e D r. C h r i s t o p h e r L a r b i e

20 ΔG = 2.303RT(pHN -php) + ZFΔψ let ΔpH = (phn -php) ΔG = 2.303RTΔpH + ZFΔψ Ζ = 1; F = 96.4 kj/mol. V ΔG = RTΔpH kj/mol. V Δψ This free energy is called proton-motive force. Actively respiring mitochondria have ΔpH = 0.75 ph units; Δψ 0.17 V; T=298 o K, R = kj/mol. K ΔG = 2.303(2.478kJ/mol)(0.75) kj/mol. V (0.17 V) = 20.6 kj/mol This is the free energy required to pump a proton across the inner mitochondrial membrane under respiring conditions. To pump 10 protons across the membrane ΔG = 206 kj. For NADH (10H + P/2e - ) ΔG o = 219 kj. All but 13 kj/mol of the free energy is used to actively transport the protons across the membrane. Similarly for FADH2 (6H + P/2e - ) ΔG o = kj/mol To pump six protons across the membrane ΔG = 6 X 20.6 kj = 124 kj. All but 33.5 kj of the free energy is used to actively transport the protons across the membrane. ATP Synthase How is the concentration gradient of protons across the inner mitochondrial membrane used to generate ATP? The proton motive force drives the synthesis of ATP as protons flow back from the intermembrane space to the matrix of the mitochondria. The protons are channelled through an enzyme call ATP synthase which catalyses the following reaction. ADP + Pi + nh + P ATP + H2O + nh + N. 20 P a g e D r. C h r i s t o p h e r L a r b i e

21 Within the mitochondrial membrane, there is a complex of proteins that carries out ATP synthesis called ATP synthase or F1F0-ATPase (named for the reverse reaction it catalyses). ATP synthase is composed of two principle complexes, the F1 unit which catalyses the synthesis of ATP. This F1 unit is associated with an integral membrane protein aggregate, the F0 unit. The F0 unit forms a transmembrane pore through which protons are channelled to drive ATP synthesis. The F1 unit is composed of five polypeptide chains α,β,γ,δ, and ε which a stoichiometry of α3β3γδε. The α and β subunits are homologous to each other, each subunit contains an ATP binding site. The catalytic sites are located in the 3 β subunits. The Structure of ATP synthase Shown in below is a side view of the F1 unit. It contains 3 α subunits and 3 β subunits arranged like the segments of an orange. The γ subunit forms a shaft. The single γ subunit associates primarily with one of the αβ pairs forcing each of the β subunits into a different conformation. One β subunit has an ADP bound, the next β-subunit contains ATP, the next is empty. This difference in nucleotide binding among the three β subunits is critical to the mechanism of the complex. 21 P a g e D r. C h r i s t o p h e r L a r b i e

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23 Shown above is the side view of the F1F0 structure. The F1 complex is in purple and grey and the F0 complex is shown is shades of yellow and red. To the immediate left is ATP synthase viewed from the P-face towards the N face. The F0 subunit is composed of three subunits denoted a, b and c in stoichiometry of ab2c The c subunits are the alpha helices that span the membrane with a small extending out into the matrix side of the membrane. The c subunits are arranged into two concentric circles with a 55Å diameter. The ring of c subunits forms a rotor that turns with respect to the a-subunit. The 2 b-subunits of F0 complex associate firmly with the α and β subunits of F1 holding them fixed relative to the membrane. In the membrane embedded cylinder (subunit c) of F0 is attached the shaft composed of the γ and ε subunits of F1. As protons flow through the membrane from the P side to the N side through the F0 channel the c subunits turn which in turn turns the embedded shaft which rotates causing the β- subunits to change conformation as the γ subunit turns. 23 P a g e D r. C h r i s t o p h e r L a r b i e

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25 In the presence of a proton gradient, ATP synthase catalyses the following reaction: ADP +Pi + E [E. ADP. Pi] [E. ATP] E + ATP In the absence of a proton gradient, there is no net synthesis of ATP, but ATP synthase catalyses the exchange of the hydroxyl groups of inorganic phosphate with the aqueous solvent as shown by the incorporation of O 18 water into phosphate shown below. 25 P a g e D r. C h r i s t o p h e r L a r b i e

26 This shows that ATP synthesis does not require the input of energy, but the release of the newly synthesized ATP does require energy. The movement of protons through the F0 channel causes the γ subunit to rotate which drives a conformational change in the structure of the β-subunit resulting in the binding of substrates (ADP and Pi) and the release of the product ATP. 26 P a g e D r. C h r i s t o p h e r L a r b i e

27 The F1 complex of ATP Synthase has three interacting conformations and three conformationally distinct active sites. The Open conformation is inactive and has a low affinity for ligands. The L form has a loose affinity for ADP and Pi and also inactive. The tight conformation is active and has a high affinity for ADP and Pi. The synthesis of ATP is initiated by the binding of ADP and Pi to an open L site. In the next step, an proton driven conformational change converts the L conformation into the T conformation and simultaneously converts the O form to the L form and the T form to the O form. In the third step ATP is synthesized at the T site and ATP is released from the O site. This cycle continues over and over. Inhibitors and Uncouplers of Oxidative Phosphorylation The electron transport chain was determined by studying the effects of particular inhibitors. Rotenone is a common insecticide that strongly inhibits the electron transport of complex I. Rotenone is a natural product obtained from the roots of several species of plants. Tribes in certain parts of the world beat the roots of trees along riverbanks to release rotenone into the water which paralyzes fish and makes them easy prey. 27 P a g e D r. C h r i s t o p h e r L a r b i e

28 Amytal is a barbiturate that inhibits the electron transport of complex I. Demerol is painkiller that also inhibits complex I. All three of these complex I inhibitors block the oxidation of the Fe-S clusters of complex I. 2-Thenoyltrifluoroacetone and carboxin specifically block electron transport in Complex II. Antimycin A1 is an antibiotic that inhibits electron transfer in complex III by blocking the transfer of electrons between Cyt bh and coenzyme Q bound at the QN site. Cyanide, azide and carbon monoxide all inhibit electron transport in Complex IV. They all inhibit electron transfer by binding tightly with the iron coordinated in Cyt a3. Azide and cyanide bind to the iron when the iron is in the ferric state. Carbon monoxide binds to the iron when it is in the ferrous state. Cyanide and azide are potent inhibitors at this site which accounts for the acute toxicity. Carbon monoxide is toxic due to its affinity for the haem iron of haemoglobin. Animals carry many molecules of haemoglobin, therefore it takes a large quantity of carbon monoxide to die from carbon monoxide poisoning. Animals have relatively few molecules of Cyt a3. Consequently an exposure to a small quantity of azide or cyanide can be lethal. The toxicity of cyanide is solely from its ability to arrest electron transport. 28 P a g e D r. C h r i s t o p h e r L a r b i e

29 Inhibitors of ATP Synthase DCCD forms covalent bonds to a glutamate residue of the c subunit of F0. When DCCD is covalently attached it blocks the proton channel, which causes the rotation and ATP synthesis to cease. Oligomycin binds directly to ATP synthase F0 subunit and blocks the flow of protons through the channel. The coupling between electron transport and oxidative phosphorylation depends on the impermeability of the inner mitochondrial membrane to H + translocation. The only way for protons to go from the intermembrane space to the matrix is through ATP synthase. Uncouplers uncouple electron transport from oxidative phosphorylation. They collapse the chemiosmotic gradient by mitochondrial membrane. 29 P a g e D r. C h r i s t o p h e r L a r b i e

30 All of the uncouplers shown below, collapse the ph gradient by binding a proton on the acidic side of the membrane, diffusing through the inner mitochondrial membrane and releasing the proton on the membranes alkaline side. 2,4-Dinitrophenol, dicumarol and carbonyl cyanidep- trifluorocarbonyl-cyanide methoxyphenyl hydrazone (FCCP) all have hydrophobic character making them soluble in the bilipid membrane. All of these decouplers also have dissociable protons allowing them to carry protons from the intermembrane space to the matrix which collapses the ph gradient. 30 P a g e D r. C h r i s t o p h e r L a r b i e

31 The potential energy of the proton gradient is lost as heat. The link between electron transport and ATP synthesis is below. (a) In the presence of excess phosphate and substrate and intact mitochondria, oxygen is consumed only when ADP is added. When all of the added ADP has been converted into ATP, electron transport stops and oxygen consumption ceases. (b) The addition of 2,4- dintrophenol uncouples electron transfer from ATP synthesis. The oxygen is completely consumed in the absence of ADP. Endogenous Uncouplers Enable Organisms to Generate Heat The uncoupling of oxidative phosphorylation from electron transport generates heat. Hibernating animals and newborn animals (including human beings) contain brown adipose tissue. The adipose tissue is brown due to the high mitochondria content of the tissue. An endogenous protein called thermogenin uncouples ATP synthesis from electron transport by opening up a passive proton channel (UCP-1) through the inner mitochondrial membrane. The collapse of the ph gradient generates heat. P/O Ratios The P/O ratio is the number of ATP molecules formed per 2 electrons transferred to oxygen. Beginning with NADH, two electrons are transferred via the electron transport chain to oxygen resulting in 10 protons being pumped across the mitochondrial inner membrane. 4 protons are channelled back the matrix per ATP synthesized. Beginning with FADH2, 6 protons pumped per 2 electrons transferred. Still 4 protons/atp. P/O ratio = 6/4 = P a g e D r. C h r i s t o p h e r L a r b i e

32 Proton Motive Force Drives Transport The primary purpose of the proton gradient is to generate ATP by oxidative phosphorylation. The potential energy of the gradient can also be used for active transport. The inner mitochondrial membrane is impermeable to charged molecules. ATP-ADP Translocase There are two specific systems to transport ADP and Pi into the mitochondrial matrix. A specific transport protein ATP-ADP translocase enables ATP and ADP to transverse the inner mitochondrial membrane. The transport of ADP in and ATP out are coupled. ADP only enters the matrix if ATP exits or vice versa. ATP-ADP translocase has a single nucleotide binding site which binds ADP and ATP with equal affinity. Due to the negative electrostatic charge of the matrix, ATP 4- is bound on the N phase of the membrane because it has greater negative charge than 32 P a g e D r. C h r i s t o p h e r L a r b i e

33 ADP 3-. Hence ATP is transported from the matrix to the intermembrane space 30 times more rapidly than ADP is transported. Once an ATP has been exported into the intermembrane space, a new molecule of ADP must be bound to the transport protein. This precisely couples the exchange of ATP for ADP in the matrix. The net result of the translocation is the decrease of the proton motive force by the net exchange of one negative charge out the matrix. The ATP-ADP translocase can be specifically inhibited by a low concentration of atractyloside which is a plant glycoside. Atractyloside binds to the translocase when its nucleotide binding site is facing the intermembrane space. This inhibitor completely blocks ATP-ADP translocation and stops oxidative phosphorylation. ATP-ADP translocase is an example of an antiporter. Other Mitochondrial Transporters Phosphate Carrier Protein An example of a symport carrier is the electronuetral symport of inorganic phosphate with a proton by the phosphate carrier protein. The combined activity of ATP-ADP translocase and the phosphate carrier is the exchange of ADP + Pi for ATP at the cost of one proton transported from the P-phase to the N-phase. 33 P a g e D r. C h r i s t o p h e r L a r b i e

34 Secondary active transport Dicarboxylate Carrier Protein This protein enables malate, succinate and fumarate to be transported from the matrix of the mitochondria in exchange for phosphate or vice versa. This is an example of anitport. Tricarboxylate Carrier Protein This proten antiports citrate and H + in exchange for malate. Pyruvate Carrier Protein Symports pyruvate and a proton from the intermembrane to the matrix. Electron Shuttle Systems Most of the NADH used in electron transport is produced in the mitochondrial matrix. The binding site of Complex I for NADH is on the matrix side of the inner membrane. The inner mitochondrial membrane is impermeable to NAD + and NADH. NADH formed in the cytosol of cells needs to reoxidized into NAD + to keep glycolysis going. Under anaerobic conditions, NAD + is regenerated by lactate dehydrogenase. Under aerobic conditions, the electrons of NADH need to be shuttled into the matrix of the mitochondria. The Glycerophosphate Shuttle This shuttle system uses two distinct glycerol 3-phosphate dehydrogenases. The first is found in the cytoplasm, the other is found on the intermembrane side of the inner mitochondrial membrane. In the first step, NADH produced in the cytosol transfers its electrons to dihydroxyacetone phosphate to form glycerol-3-phosphate. Glycerol-3- phosphate enters the inter-mitochondrial space through a porin. Glycerol-3-phosphate is then reoxidized into dihydroxyacetone phosphate by an FADdependent 34 P a g e D r. C h r i s t o p h e r L a r b i e

35 mitochondrial membrane glycerol 3- phosphate dehydrogenase. In this shuttle the electrons of NADH are transferred to FAD to form FADH2. The two electrons bound by the FADH2 are transferred directly to coenzyme Q forming QH2. QH2 carries the electrons to complex III. The result of this shuttle is 1.5 ATP/NADH. Note that this shuttle is essentially irreversible. The Malate-Aspartate Shuttle In the cytosol, oxaloacetate is reduced to malate by malate dehydrogenase which uses NADH as the reductant. Malate is transported across the inner mitochondrial membrane by the dicarboxylic acid or tricarboxylic acid carrier. Now in the matrix, the malate is reoxidized by malate dehydrogenase to generate oxaloacetate and NADH which can now transfer its electrons to Complex I. 35 P a g e D r. C h r i s t o p h e r L a r b i e

36 The oxaloacetate is transaminated by glutamine to form aspartate and α-ketoglutarate. Aspartate can be transported across the inner mitochondrial membrane by the dicarboxylic acid carrier. In the cytosol aspartate transaminates α-ketoglutarate to reform oxaloacetate completing the cycle. This shuttle system generates 2.5 ATP/NADH and is completely reversible. The net yield of ATP by glucose oxidation depends on the shuttle used. Glycolysis: Glucose 2 pyruvate + 2ATP + 2NADH Back to the very beginning. Glucose + 6O2 6CO2 + 6H2O ΔG = 2937 kj/mol Under cellular conditions, ΔG for ATP hydrolysis 50 kj/mol 32 ATP /glucose = 1,600 kj/mol Efficiency 1600/2937 X 100 = 54% 3.5 billion years of evolution has resulted in 54 % efficiency. OXYGEN METABOLISM AND TOXICITY Chemiosmotic Theory Chemiosomotic Theory state that the free energy of electron transport is coupled to the pumping of protons from the matrix to the intermembrane space to create a ph gradient. This potential energy stored in this ph gradient is used to drive the synthesis of ATP. This process requires: 1. An intact inner mitochondrial membrane that is impermeable to protons, 2. The inner mitochondrial membrane must also be impermeable to ions such as OH -, K +, Na + and Cl - because free diffusion of these ions would discharge the trans-membrane electrical potential which is a key component of the proton motive force. 36 P a g e D r. C h r i s t o p h e r L a r b i e

37 3. Compounds that increase the permeability of the membrane to protons dissipate the electrochemical gradient and uncouple electron transport from oxidative phosphorylation. Ca 2+ transport in the mitochondria There are two separate transport proteins for calcium ions. One integral membrane protein transport calcium ions into the matrix using the trans-membrane electrical potential. The rate of Ca 2+ influx depends on the cytosol concentration of calcium because the Km for Ca 2+ for this transport protein is greater than physiological cytosolic concentrations of Ca 2+. A separate protein transports Calcium ions out of the matrix antiporting in a sodium ion. This transporter protein always operates at its maximal velocity. Thus an increase in cystolic Ca 2+ increases the rate of influx while the rate of efflux remains unchanged. This produces a net increase in the concentration of Ca 2+ in the matrix. A drop in cystolic Ca 2+ cause a decrease in the rate of influx while the rate of efflux remains unchanged thus producing a net drop in the concentration of Ca 2+ in the matrix. Cool. Regulation of ATP Producing Pathways The electron transport chain functions near equilibrium from NADH to cytochrome c. There is only one irreversible step in electron transport-oxidative phosphorylation. That is the reduction of oxygen by cyctochrome c oxidase, Complex IV. Cytochrome c oxidase is regulated primarily by the concentration of reduced cytochrome c. The concentration of reduced cytochrome c is in equilibrium with the rest of the electron transport pathway. Thus a high NADH/NAD + ratio and a low ATP/ADP ratio increase the concentration of reduced Cyt c. Increased concentration of reduced Cyt c results in an increase in the rate of oxygen reduction. 37 P a g e D r. C h r i s t o p h e r L a r b i e

38 Oxygen Metabolism Molecular oxygen has two unpaired electrons which have parallel spin states. The parallel spin states prevent carbon based organisms such as us from spontaneously igniting in our oxygen atmosphere. The parallel electron spins prevent oxidation by 2 electron transfers. Oxidation by molecular oxygen can only occur by the transfer of single electrons. Organic molecules that serve as substrates for oxidation do not contain unpaired electrons. Their bonds are in the stable form of two electrons with antiparallel spins. For O2 to accept a pair of electrons from an organic substrate, one of the electrons of oxygen or one of the electrons from the donating substrate has to invert its spin. There is a large thermodynamic barrier to such spin inversions. As a result two electron oxidations by molecular oxygen have to occur stepwise by two single electron transfers. The large barrier to spin inversions keeps us from spontaneously combusting in our oxygen atmosphere. The two unpaired electrons of molecular oxygen have a biradical nature which has a tendency to form highly reactive oxygen species (ROS) which initiate free radical reactions. Free radicals react indiscriminately with any molecule they come in contact with. Free radical reactions are a chain of single electron transfer reactions which damage cellular components. Reactive Oxygen Species (ROS). OH Hydroxyl radical. O2 - Superoxide H2O2 Hydrogen peroxide Reactive oxygen species can be lethal to cells. Proteins, membrane lipids, carbohydrates and nucleic acids are subject to cellular damage by oxygen radicals. Free radical damage contributes to complications of many chronic diseases. In some cases oxygen free radicals are the direct cause of the disease state. In other cases such as rheumatoid arthritis, radical oxygen species perpetuate cellular damage caused by a different process. Macrophages, neutrophils use ROS to destroy foreign organisms during phagocytosis. 38 P a g e D r. C h r i s t o p h e r L a r b i e

39 Free radical mediated cellular injury Superoxide and hydroxyl free radicals initiate peroxidation in the cellular, mitochondrial, nuclear, and endoplasmic reticulum membranes. This increases the cellular permeability for Ca 2+. Increased cellular concentrations of calcium ions damage the mitochondria. Amino acids are oxidized and degraded. Nuclear and mitochondrial DNA is oxidized, resulting in strand breaks and other types of DNA damage. Coenzyme Q generates superoxide One of the major sites of superoxide generation is the electron transport chain which leaks free radicals in the form of semiquinone radicals of coenzyme Q. The one electron form of CoQ occasionally leaks into the inner mitochondrial membrane. The nonspecific interaction of a CoQH. with molecular oxygen results in the formation of a superoxide radical which abstracts an electron from some other molecule and initiates a free radical chain reaction Cellular Defences Against ROS 39 P a g e D r. C h r i s t o p h e r L a r b i e

40 Cells protect themselves by compartmentalizing processes which generate highly reactive oxygen species. Oxidative stress occurs when the rate of generation of ROS exceeds the capacity of the cell to remove them. Aerobic cells have to protect themselves from damage by the naturally occurring, continuous generation of ROS. Superoxide dismutase (SOD) removes the superoxide free radical. Superoxide dismutatase is one of the primary defenses against oxidative stress because the superoxide radical is a strong free radical initiator. Catalase and glutathione peroxidase removes hydrogen peroxide and lipid peroxides. Hydrogen peroxide is a source of hydroxyl free radicals. Catalase reduces hydrogen peroxide into water. Catalase is found in the peroxisomes. Glutathione peroxidase also protects the cell from oxidative injury by reducing hydrogen peroxide into water and lipid peroxides into acids. The mitochondria is a major source of ROS. The mitochondria have a high concentration of SOD and glutathione peroxidase to prevent oxidative stress. Vitamins E and C act as antioxidants Vitamin E (tocopherol) is the most abundant antioxidant in nature. It is a lipophilic free radical scavenger to protect lipids from peroxidation in the membranes. It sole biological purpose is to quench free radical reactions in membranes. 40 P a g e D r. C h r i s t o p h e r L a r b i e

41 Vitamin C, ascorbic acid is a water soluble free radical scavenger. It accepts electrons from superoxide, hydrogen peroxide, hypochlorite, hydroxyl radicals and peroxyl radicals. It also quenches ozone and nitrous oxide. The role of oxygen in cell injury Oxygen is necessary for the generation of ATP by oxidative phosphorylation. But oxygen is also toxic. Normal oxygen metabolism generates reactive oxygen species which can cause cell injury. Protective enzymes and antioxidants remove ROS. Various stimuli, such as radiation, inflammation, aging and high concentrations of oxygen greatly increase the rate of formation of ROS. Ozone and nitrous oxide are air pollutants that form free radicals in the cells of the lungs resulting in pulmonary emphysema and 41 P a g e D r. C h r i s t o p h e r L a r b i e

42 pulmonary fibrosis. The lack of oxygen due to decreased blood flow (ischemia) also causes cell injury. The reintroduction of oxygen (reperfusion) enhances cellular injury due to ROS. Hypoxic Injury Hypoxia- deficiency of oxygen. Hypoxic injury-injury caused by a deficiency of oxygen. Acute hypoxic tissue injury has been extensively studied. The occlusion of a major coronary artery produces an array of biochemical and physiological complications. When a tissue is deprived of oxygen, the mitochondrial electron transport-oxidative phosphorylation is inhibited, resulting in a severe decline in cellular ATP levels. Anaerobic glycolysis is activated in an attempt to restore cellular ATP levels. Glycogen stores are rapidly depleted, and lactic acid concentrations increase, lowering the cellular ph (lactic acidosis). Hypoxic cells begin to swell as they can no longer maintain their normal electrolyte concentrations. The mitochondria begin to swell and accumulate calcium which precipitates as calcium phosphate. As the membranes swell they become permeable leading to the leakage of enzymes, coenzymes and other cellular constituents. As the ph continues to fall, the lysosomal membranes release hydrolytic proteases, lipases, glucosides and phosphatases that digest the cell. Amazingly cells that have been exposed to short periods of hypoxia can recover without irreversible damage upon reperfusion with oxygen containing medium. 42 P a g e D r. C h r i s t o p h e r L a r b i e

43 Enzymes that use oxygen as a substrate The electron transport system of the mitochondria accounts for 90% of the oxygen consumption of a cell. The remainder 10 % of the oxygen is consumed in oxygen requiring reactions in the body by oxidases or oxygenases. Oxidases An oxidase is an enzyme that reduces molecular oxygen to water or hydrogen peroxide. These oxidases play specific roles in metabolism. Oxidases reduce oxygen to either water or hydrogen peroxide. Cytochrome c oxidase (Complex IV) is an example of an oxidase. This enzyme complex catalyses the four electron reduction of O2 to 2H2O. The electrons are transferred to oxygen one at a time to overcome the spin restrictions. The mechanism of this enzyme allows the peroxy intermediates to formed in a controlled fashion without interaction between oxygen free radicals and other mitochondrial components. Other oxidases reduce oxygen to peroxide instead of water. These enzymes are compartmentalized into peroxisomes and lysosomes where catalase and glutathione peroxidase remove the hydrogen peroxides. Oxygenases An oxygenase directly incorporates oxygen into the molecules being oxidized. Oxygenases incorporate oxygen into the substrate. Monooxygenases incorporate one of the atoms of oxygen into the substrate while reducing the other atom of oxygen into water. The common name for monooxygenases is hydroxylases (i.e. phenylalanine hydroxylase which adds a hydroxyl group to phenylalanine to form tyrosine.) Monooxygenases require an electron donor such as NADPH, a coenzyme capable of mediating between a two electron donor and a one electron acceptor (FAD) and a metal cofactor to form a stable reactive oxygen complex. Cytochrome P450 enzymes are a superfamily of structurally related monooxygenases which hydroxylate many compounds such as steroids, fatty acids, drugs, carcinogens, etc. There are over 100 isozymes of P450 enzymes in humans, each with a different but overlapping specificity. Dioxygenases incorporate both atoms of oxygen into the substrate. Pyruvate Dehyrogenase Deficiency A variety of pyruvate metabolism disorders have been detected in children. Some involve deficiencies in the regulatory subunits of pyruvate dehydrogenase other disorders involve the catalytic subunits. Children with deficiencies have high serum concentrations of lactate, pyruvate and alanine which produce a chronic lactic acidosis. In addition there are severe neurological defects that in most cases result in death. Patients in shock suffer from lactic acidosis because of the decreased delivery of oxygen 43 P a g e D r. C h r i s t o p h e r L a r b i e

44 increases anaerobic glycolysis. Decreased concentrations of oxygen increase mitochondrial concentrations of NADH which activates pyruvate dehydrogenase kinase which inhibits pyruvate dehyrdogenase. Patients in shock are treated with dichloroacetate which is an inhibitor of pyruvate dehydrogenase kinase and therefore an activator of pyruvate dehydrogenase. Fumarase Deficiency Deficiencies in the enzymes of the citric acid cycle are rare because these enzymes are so crucial for life. Several cases are now on record in which there is a severe deficiency in fumarase in both the mitochondria and the cytosol of tissues. The condition is characterized by encephalopathy (a disease of the brain involving alteration of brain structure.), severe impairment of neurological function and dystonia (disordered tonicity of muscles). These conditions develop immediately after birth. The urine contains abnormally high concentrations of fumarate, succinate, citrate and malate. Mitochondrial Myopathies Mitochondrial myopathies are mitochondrial diseases that affect the muscles. Clinically, patients with mitochondrial myopathies complain of weakness and severe cramping of the affected muscles. Infants have difficulty feeding and crawling. Continued severe fatigue results in minimal exertion and muscle wasting. Examining the mitochondria of these myopathies have discovered primary reasons for mitochondrial malfunction. 1. Deficiencies in mitochondrial transport. 2. Deficiencies in electron transport in either Complex I, Complex II, Complex III, Complex IV and ATP synthase have been described. 3. Crystalline inclusions within the mitochondrial matrix. Under these conditions electron transport and oxidative phosphorylation are only loosely coupled. Example of paracrystalline inclusions in the mitochondria from muscles of ocular myopathic patients. 44 P a g e D r. C h r i s t o p h e r L a r b i e