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Inhibitors & uncouplers of oxidative phosphorylation & ETC

  1. Inhibitors & Uncouplers of OXIDATIVE PHOSPHORYLATION & ETC Dipesh Tamrakar MSc. Clinical Biochemistry 1
  2. OVERVIEW • Review • Q-cycle • Uncoupler proteins • Inhibitors of Oxidative phosphorylation and ETC 2
  3. REVIEW 3
  4. REVIEW • The mitochondrial respiratory chain consists of a series of sequentially acting electron carriers, most of which are integral proteins with prosthetic groups capable of accepting and donating either one or two electrons. • Three types of electron transfers occur in oxidative phosphorylation: 1. direct transfer of electrons, as in the reduction of Fe3+ to Fe2+; 2. transfer as a hydrogen atom (H+ + e- ); and 3. transfer as a hydride ion (:H-), which bears two electrons. • The term reducing equivalent is used to designate a single electron equivalent transferred in an oxidation- reduction reaction. 4
  5. Lipid-soluble benzoquinone with a long isoprenoid side chain UBIQUINONE (Q or Coenzyme Q) can accept one electron to become the semiquinone radical ('QH) or two electrons to form UBIQUINOL (QH2) 5
  6. • Iron associated with inorganic sulfur atoms of with sulfur atoms of Cys residues in the protein, or both • lron-sulfur centers :The Fe-S centers of iron-sulfur proteins may be as simple as: (a) with a single Fe ion surrounded by the S atoms of four Cys residues. Other centers include both inorganic and Cys S atoms as in (b) 2Fe-2S or (c) 4Fe-4S centers 6
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  9. • View of the complex shows how cytochrome c1 and the Rieske iron-sulfur protein project from the p surface and can interact with cytochrome C in the intermembrane space. 9
  10. Q cycle • The process by which electrons travel from QH2 to Cytochrome C is known as Q- cycle • The Q cycle, shown in two stages. 1. The path of electrons through Complex II is shown by blue arrows In the first stage (left) Q, on the N side is reduced to the semiquinone radical which in the second stage (right) is converted to QH2. 10
  11. 2. Meanwhile on the P side of the membrane two molecules of QH2 are oxidized to Q, releasing two protons per Q molecule (four protons in all) into the intermembrane space. Each QH2 donates one electron ( via the Rieske Fe-S center) to cytochrome cl, and one electron( via cytochrome b ) to a molecule of Q near the N side, reducing it in two steps to QH2. This reduction also uses two protons per Q, which are taken up from the matrix. 11
  12. Q-cycle 12
  13. Path of electron through complex IV • The 3 proteins critical to electron flow are subunits I, II and III. • Electron transfer through complex IV begins with cytochrome C. 2 molecules of reduced cytochrome C each donate an electron to the binuclear center CuA. • From here electrons pass through heme a to the Fe-Cu center (cytochrome a3 and CuB) • Oxygen now binds to heme a3 and is reduced to its peroxy derivative by 2 electrons from the Fe-Cu center. • Delivery of 2 more electrons from cytochrome c converts the O2- 2 to 2 molecules of water, with consumption of 4 substrate protons from the matrix. • At the same time, 4 protons are pumped from the matrix by an as yet unknown mechanism 13
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  16. Shuttle systems indirectly convey cytosolic NADH into Mitochondria for Oxidation • The NADH dehydrogenase of the inner mitochondrial membrane of animal cells can accept electrons only from NADH in the matrix • Malate-aspartate shuttle is the special shuttle system that carry reducing equivalents from cytosolic NADH into mitochondria by an indirect route • Functions mainly in liver, kidney, and heart mitochondria • The reducing equivalents of cytosolic NADH are first transferred to cytosolic oxaloacetate to yield malate, catalyzed by cytosolic malate dehydrogenase • The malate thus formed passes through the inner membrane via the malate--ketoglutarate transporter 16
  17. • Within the matrix the reducing equivalents are passed to NAD- by the action of matrix malate dehydrogenase forming NADH; this NADH can pass electrons directly to the respiratory chain • About 2.5 molecules of ATP are generated as this pair of electrons passes to 02. • Cytosolic oxaloacetate must be regenerated by transamination reactions and the activity of membrane transporters to start another cycle of the shuttle. • Skeletal muscle and brain use a different NADH shuttle, the Glycerol 3 phosphate shuttle 17
  18. Malate Aspartate shuttle 18
  19. • This alternative means of moving reducing equivalents from the cytosol to the mitochondrial matrix operates in skeletal muscle and the brain. • In the cytosol, dihydroxyacetonephosphate accepts two reducing equivalent from NADH in a reaction catalyzed by cytosolic glycerol 3 –phosphate dehydrogenase. • An isozyme of glycerol3 –phosphate dehydrogenase bound to the outer face of the inner membrane then transfers two reducing equivalents from glycerol3 –phosphate in the intermembrane space to ubiquinone. • Note that this shuttle does not involve membrane transport systems. 19
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  21. • Substrate shuttles for the transportof electrons across the inner mitochondrial membrane. A. Glycerophosphate shuttle. B. Malate-aspartate shuttle. DHAP = dihydroxyacetone phosphate; NAD(H) = nicotinamide adenine dinucleotide; FAD(H2) = flavin adenine dinucleotide; CoQ = coenzyme Q. 21
  22. Uncoupling protein • An uncoupling protein (UCP) is a mitochondrial inner membrane protein that is a regulated proton channel or transporter. • An uncoupling protein is thus capable of dissipating the proton gradient generated by NADH-powered pumping of protons from the mitochondrial matrix to the mitochondrial intermembrane space. • The energy lost in dissipating the proton gradient via UCPs is not used to do biochemical work. Instead, heat is generated. • This is what links UCP to thermogenesis. • UCPs are positioned in the same membrane as the ATP synthase, which is also a proton channel. 22
  23. • The two proteins thus work in parallel with one generating heat and the other generating ATP from ADP and inorganic phosphate, the last step in oxidative phosphorylation. • Mitochondria respiration is coupled to ATP synthesis (ADP phosphorylation) but is regulated by UCPs. • There are five types of homologs known in mammals: • UCP1, also known as thermogenin • UCP2 • UCP3 • SLC25A27, also known as "UCP4" • SLC25A14, also known as "UCP5" 23
  24. • Uncoupling proteins play a role in normal physiology, as in cold exposure or hibernation, because the energy is used to generate heat instead of producing ATP. • Some plants species use the heat generated by uncoupling proteins for special purposes. • Skunk cabbage, for example, keeps the temperature of its spikes as much as 20° higher than the environment, spreading odor and attracting insects that fertilize the flowers. • However, other substances, such as 2,4-dinitrophenol and carbonyl cyanide m-chlorophenyl hydrazone, also serve the same uncoupling function, and are considered poisonous • Salicylic acid is also an uncoupling agent and will decrease production of ATP and increase body temperature if taken in excess. • Uncoupling proteins are increased by thyroid hormone, norepinephrine, epinephrine, and leptin. 24
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  26. Inhibitors of Electron Transport: • These are the inhibitors that arrest respiration by combining with members of the respiratory chain, rather than with the enzymes that may be involved in coupling respiration with ATP synthesis. • They appear to act at 3 loci that may be identical to the energy transfer sites I, II and III. The given below are the inhibitors of Electron transport chain. 26
  27. Rotenone • It inhibits the transfer of electrons from iron-sulfur centers in complex I to ubiquinone. • This interferes with NADH during the creation of usable cellular energy (ATP) • Complex I is unable to pass off its electron to CoQ, creating a back-up of electrons within the mitochondrial matrix. • Cellular oxygen is reduced to the radical, creating reactive oxygen species, which can damage DNA and other components of the mitochondria • It is the non-toxic inhibitors of Electron transport chain. • This is non-toxic to mammals because poorly absorbed. Shows toxic effect in fishes. 27
  28. Piericidin A: • It is an Antibiotic. • It is produced by species of streptomyces. • The action is similar to Rotenone. Barbiturates (Amytal, Seconal): • It blocks NADH dehydrogenase and Coenzyme.Q Antimycins: • These are antibiotic, produced by Streptomyces. One of the inhibitor in ETC. • It inhibits around site II and block electron flow between cytochromes b and c1, which prevents ATP synthesis coupled to the generation of a proton gradient at site II. • About 0.07 micromole of antimycin A per gram of mitochondrial protein is effective. 28
  29. Dimercaprol: • It is identical in action to the antimycins. Cyanides: • The cyanide ion (CN–) combines tightly with cytochrome oxidase, leading to inhibition of ETC Azide: • Azide blocks the electron flow between the cytochrome oxidase complex and oxygen. • Azide reacts with the ferric form (Fe3 +) of this carrier. Hydrogen Sulfide: • H2S is toxic, with disagreeing odour gives warning. • It inhibits Cytochrome Oxidase. Carbon Monoxide: • It blocks between cytochrome oxidase and Oxygen. • It inhibits Fe2 + 29
  30. Inhibitors of Oxidative Phosphorylation: Oligomycins: • Is a polypeptide antibiotic are obtained from various species of “Streptomyces”. • The antibiotic is potent inhibitor to ATP synthase complex. • binds to the Fo domain of ATP synthase, closing the proton channel and preventing reentry of protons into the matrix, there by preventing phosphorylation of ADP to ATP. • Because the pH and electrical gradients cannot be dissipated in the presence of this drug, electron transport stops because of the difficulty of pumping any more protons against the steep gradients . • This dependency of cellular respiration on the ability to phosphorylate ADP to ATP is known as respiratory control and is the consequence of the tight coupling of these processes. 30
  31. Rutamycin: • This antibiotic also inhibits both ETC and oxidative phosphorylation. Atractylate: • It backs oxidative phosphorylation by compelling with ATP & ADP for a site on the ADP-ATP antiport of the mitochondrial membranes. One of the inhibitors list which blocks the oxidative phosphorylation. Bongkrekate: • It is a toxin formed by bacteria (Pseudomonas) in a coconut preparation from Java. • It also blocks the ADP-ATP antiport. 31
  32. Uncouplers of Oxidative Phosphorylation: • Uncouplers can be defined as A substance that uncouples phosphorylation of ADP from electron transfer. • Uncoupling agents are compounds which dissociate the synthesis of ATP from the transport of electrons through the cytochrome system. • This means that the electron transport continues to function, leading to oxygen consumption but phosphorylation of ADP is inhibited. 32
  33. 2,4-Dinitrophenol: • A classic uncoupler of oxidative phosphorylation. • was used as a weight-loss agent in the 1930s • The substance carries protons across the inner mitochondria membrane. • In the presence of these uncouplers, electron transport from NADH to O2 proceeds normally, but ATP is not formed by the mitochondria. • Body temperature is elevated as a result of hyper metabolism. • When phosphorylation is uncoupled from electron flow, a decrease in the proton gradient across the inner mitochondrial membrane and, therefore , impaired ATP synthesis is expected. • In an attempt to compensate for this defect in energy capture , metabolism and electron flow to oxygen is increased. • This hyper metabolism will be accompanied by elevated body temperature be cause the energy in fuels is largely wasted, appearing as heat. 33
  34. Dicoumarol (Vitamin.K analogue): • Used as anticoagulant. Calcium: • Transport of Ca+2 ion into mitochondria can cause uncoupling. • Mitochondrial transport of Ca+2 is energetically coupled to oxidative phosphorylation. • It is coupled with uptake of pi • When calcium is transported into mitochondria, electron transport can proceed but energy is required to pump the4 Ca+2 into the mitochondria. Hence, no energy is stored as ATP. CCCP (Chloro carbonyl cyanide phenyl hydrazone): • Most active uncoupler • These lipid soluble substances can carry protons across the inner mitochondrial membrane. 34
  35. Valinomycin: • This is the example to Ionophore of oxidative phosphorylation. • Produced by a type of streptomyces • It is a repeating macrocyclic molecule made up of four kinds of residues (L-lactate, L-Valine, D-hydroxyisovalarate and D- Valine) taken 3 times. • Transports K+ from the cytosol into matrix and H+ from matrix to cytosol, thereby decreasing the proton gradient. Physiological un-couplers: • Excessive thyroxin hormone • EFA deficiency • Long chain FA in brown adipose tissue • Unconjugated hyperbilirubinaemia 35
  36. Uncoupling Proteins and the Molecular Mechanisms of Thyroid Thermogenesis • TH is synthesized in the thyroid gland and controlled by thyroid peroxidase activity that regulates the iodination, coupling, and ultimately proteolysis of tyrosine residues on thyroglobulin to release the THs, T4 and T3, into the bloodstream • The lesser active T4 is released from the thyroid gland at higher concentrations than the more active T3 and is locally converted to T3 in target tissues by the actions of tissue- specific deiodinases • Two genes, THRA and THRB, are responsible for the expression of distinct thyroid hormone receptors (TRs), each of which are alternatively spliced to produce multiple isoforms, TRα1, TRα2, TRβ1, and TRβ2, respectively. 36
  37. • With the exception of TRα2, which does not bind T3 and functions to repress T3 actions, TR isoforms mediate distinct functions (both stimulatory and repressive) in response to and in the absence of T3. • Integral to their functions as transcriptional regulators, TRs bind other nuclear hormone receptors, coactivators, and corepressors, the details of which have been reviewed elsewhere . • In addition to its transcriptional regulation, recent work has also revealed that TH may regulate cell signaling pathways non-genomically. • However, it is not yet established whether and how TH may influence body temperature apart from its role as a ligand for thyroid hormone receptor-dependent gene transactivation 37
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  39. Thank-you 39

Notes de l'éditeur

  1. NADH:ubiquinone oxidoreductase (Complex l ). Complex I catalyzes the transfer of a hydride ion from NADH to FMN, from which two electrons pass through a series of Fe-S centers to the ironsulfur protein N -2 in the matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses in to the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons The detailed mechanism that couples electron and proton transferin Complex I is not yet known but probably involves a Q cycle similar to that in Complex ll l in which QH2 participates twice per electron pair (see Fig.1 9-12). Proton flux produces an electrochemical potential across the inner mitochondria membrane N side negative, side positive) which conserves some of the energy released by the electron-transfer actions This electrochemical potential drives ATP synthesis.
  2. Path of electrons from NADH,succinate fatty acyl-CoA, and glycerol3 –phosphate to ubiquinone. Electrons from NADH pass through a flavoprotein to a series of iron-sulfur proteins ( in Complex l ) and then to Q. Electrons from succinate pass through a flavoprotein and several Fe-S centers ( in Complex ll) on the way to Q. Clycerol3 – phosphate donates electrons to a flavoprotein (glycerol3 –phosphate dehydrogenase) the outer face of the inner mitochondrial membrane, from which they pass to Q. Acyl-CoA dehydrogenase(the first enzyme of B oxidation) transfers electrons to electron-transferring flavoprotein (ETF) from which they pass to Q via ETF:ubiquinone oxidoreductase
  3. The complex has two distinct binding sites for ubiquinone Qn and Qp, which correspond do the sites of inhibition by two drugs that block oxidative phosphorylation. –Myxothiazol,which prevents electron flow fromQH2, to the Rieske iron-sulfur protein binds at Qp, near the 2Fe-2S center and heme b1 on the p side The dimeric structure is essential to the function of ComplexIII. The interface between monomers forms two caverns each containing a Qp site from one monomer and a Qn, site from the other. The ubiquinone intermediates move within these sheltered caverns.
  4. The larger green structure includes the other 10 proteins in the complex
  5. NADH in the cytosol passes 2 reducing equivalents to oxaloacetate, producing malate Malate crosses the inner membrane via the malate alpha ketoglutarate transporter In the matrix, malate passes 2 reducing equivalents to NAD+, and the resulting NADH is oxidized by the respiratory chain; the oxaloacetate formed from malate cannot pass directly into the cytosol Oxaloacetate is first transminated to aspartate Aspartate can leave via the glutamate-aspartate transporter Oxaloacetate is regenerated in the cytosol, completing the cycle
  6. This uncoupler cause electron transport to proceed at a rapid rate without establishing a proton gradient, much a s do the UCPs . Again, energy is released as heat rather than being used to synthesize ATP. [Note: In high doses, aspirin and other salicylates uncouple oxidative phosphorylation. This explains the fever that accompanies toxic overdoses of these drugs .]
  7. responsible for heat production in the brown adipocytes of mammals. In brown fat, unlike the more abundant white fat, almost 90% of its respiratory energy is used for thermogenesis in response to cold in the neonate and during arousal in hibernating animals. However, humans appear to have few concentrated deposits of brown fat (except in the newborn), and UCP1 does not appear to play a major role in energy balance.
  8. ATP are because the proton motive force across the inner mitochondrial membrane is dissipated The electron transport cha in will s till be inhibited by cyanide .
  9. Tissue specific mechanisms of TH-mediated thermogenesis. In response to cold exposure, NorEpinephrine released from SNS (sympathetic nervous system) nerve terminals binds β3-adrenergic receptors (β-AR) in BAT and WAT, increasing local and systemic FFA release along with an induction/activation of UCP1/3 and other potential thermogenic genes in BAT and skeletal muscle, respectively. Simultaneously, SNS stimulation activates D2 deiodinases in BAT and skeletal muscle that increase T3 levels, leading to the transactivation via TRs of thermogenic genes that ultimately govern the thermogenic capacities of BAT and skeletal muscle including PGC1α and mGPD. In BAT, UCP1 is activated by FFA release to transport protons from the mitochondrial intermembrane space (IMS) to the matrix, dissipating the proton gradient to produce heat. Similarly, we propose that skeletal muscle NST ( nonshivering thermogenesis)  is activated in part by the uptake of FFA released from SNS-stimulated WAT lipolysis and UCP3 activation. MCT8, monocarboxylate transporter 8.