The document summarizes key concepts about energy transformations that occur in the human body through respiration and oxidative phosphorylation. Chemical energy from nutrients is converted to ATP through substrate-level phosphorylation and oxidative phosphorylation. Oxidative phosphorylation uses the electron transport chain in the mitochondria to generate a proton gradient across the inner mitochondrial membrane, which drives ATP synthesis from ADP and inorganic phosphate.

1. Respiratory chain ~ Rea ctive oxygen species © Department of Biochemi stry, MU Brno (J.D.) 2011

2. The first law of thermodynamics  U =  W +  Q = work + heat less utilizable form of energy Energy can be converted from one form to another, but cannot be created or destroyed.

3. Transformation of energ y in human body chemical energy of nutrients = work + heat ener gy of nutrients = BM + phys. activity + reserves + heat energy input energy output any work requires ATP BM = basal metabolism

4. Energy transformations in the human body are accompanied with continuous production of heat chemical energy of nutrients heat NADH+H + FADH 2 proton gradient across IMM heat heat ATP 1 2 3 1 . .. metabolic dehydrogenations with NAD + and FAD 2 . . . respiratory chain (oxidation of reduced cofactors + reduction of O 2 to H 2 O) 3 . . . oxidative phosphorylation, IMM inner mitochondrial membrane 4 . . . transformation of chemical energy of ATP into work + some heat  . .. high energy systems work 4

5. Energetic data about nutrients 30 % 6 % 4 % Thermogenesis 10 % 17 Proteins 60 % 17 Saccharides 30 % SAFA 5 % , MUFA 20 % , PUFA 5 % 38 Lipids E nergy supply/day Energy (kJ/g) Nutrient

6. glucose: 6.7 % H a verage ox. num. of C = 0 . 0 alanine: 7.9 % H a verage ox. num. of C = 0 . 0 stearic acid: 12.8 % H a verage ox. num. of C = -1 . 8  C is the most r edu ced Oxidation numbers of carbon and the content of hydrogen in nutrients

7. Two ways of ATP formation in body Compare: Phosphorylation substrate-OH + ATP  substrate-O-P + ADP (e.g. phosphorylation of glucose, proteins, etc., catalyze kinases) higher energy content than ATP 2. S ubstrate-level phosphorylation ( ~ 5 % ATP) ADP + macroergic phosphate-P  ATP + second product 1. O xidative phosphorylation in the presence of O 2 ( ~ 95 % ATP ) ADP + P i + energy of H + gradient  ATP

8. Distinguish ! consumed Phosphorylation of a substrate produced Substrate-level phosphorylation produced Oxidative phosphorylation ATP is Process

10. Ph os ph oryla tion of GDP in citrate cycle guanosine diphosphate ATP succinate guanosine triphosphate succinyl-CoA succinyl phosphate is made from succinyl-CoA + P i

11. Ph os ph oryla tion of ADP by 1,3-bis ph os ph oglycer ate 1,3-bisP-glycerate 3-P-glycerate

12. Ph os ph oryla tion of ADP by ph os ph oenolpyruv ate pyruvate enolpyruvate phosphoenolpyruvate

13. Aerobic phosphorylation follows the re oxida tion of redu ced c ofa ctors in R.CH. Nutrients (redu ced form s of C) CO 2 + redu ced c ofa c tor s (NADH+H + , FADH 2 ) reoxida tion in R.CH. dehydrogena tion O 2 Proton gradient + H 2 O ADP + P i  ATP de carboxylation

18. Aspart a t e /mal ate shuttle malate malate oxal o acetate Inner mitochondrial membrane oxal o acetate cytoplasm hydrogenation dehydrogenation transamination MD malate dehydrogenase AST aspartate aminotransferase

19. Glycerol ph os phate shuttle glycerol 3-P dihydroxyacetone phosphate Inner mitochondrial membrane GPD = glycerol 3-P dehydrogenase ubiquinol ubiquinone

20. Respiratory chain is the system of redox reactions in IMM which starts by the NADH oxidation and ends with the reduction of O 2 to water . The free energy of oxidation of NADH/FADH 2 is utilized for pumping protons to the outside of the inner mitochondrial membrane. The proton gradient across the inner mitochondrial membrane represents the energy for ATP synthesis . H + H + H + H + – – – + + + ADP + P i ATP NAD H + H + O 2 2H 2 O NAD + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + e – proton gradient M atrix (negativ e) IMS (po sitive)

22. Coenzyme FMN (as well as FAD) transfers two atoms of hydrogen . Flavoproteins contain flavin prosthetic group as flavin mononucleotide (FMN, complex I) or flavin adenine dinucleotide (FAD, complex II): N N N NH O O H 3 C N N N N O O H H + 2 H – 2 H FMN oxidized form FMN H 2 reduced form H 3 C H 3 C H 3 C CH 2 –O– P CH 2 H–C–OH H–C–OH H–C–OH CH 2 –O– P CH 2 H–C–OH H–C–OH H–C–OH

23. Iron-sulfur proteins (FeS-proteins, non-heme iron proteins) Despite the different number of iron atoms present, each cluster accepts or donates only one electron . Fe 2 S 2 cluster Fe 4 S 4 cluster

24. It accepts stepwise two electrons (one from the complex I or II and the second from the cytochrome b ) and two protons (from the mitochondrial matrix), so that it is fully reduced to ubiquinol: Ubiquinone ( coenzyme Q ) The very lipophilic polyisoprenoid chain is anchored within phospholipid bilayer. The ring of ubiquinone or ubiquinol (not semiquinone) moves from the membrane matrix side to the cytosolic side and translocates electrons and protons. R = –(CH 2 –CH=C–CH 2 ) 10 -H CH 3 + e + H + ubiquin one , Q semi quinone, •QH ubiquin ol , QH 2 + e + H +

25. Cytochromes are heme proteins , which are one-electron carriers due to reversible oxidation of the iron atom: Mammalian cytochromes are of three types – a , b , c . They differ in the substituents attached to the porphin ring. All these types of cytochromes occur in the mitochondrial respiratory chain. Cytochromes type b (including cytochromes class P-450) occur also in membranes of endoplasmic reticulum and the outer mitochondrial membrane. N N N N F e 2+ N N N N F e 3+ + e – – e –

26. Some differences in cytochrome structures Cytochrome c central Fe ion is attached by coordination to N-atom of His 18 and to S-atom of Met 80 ; two vinyl groups bind covalently S-atoms of Cys 14 + Cys 17 . The heme is dived deeply in the protein terciary structure so that it is unable to bind O 2 , CO . Cyt c is water-soluble, peripheral protein that moves on the outer side of the inner mitochondrial membrane. Cytochrome aa 3 central Fe ion is attached by coordination to two His residues; one of substituents is a hydrophobic isoprenoid chain, another one is oxidized to formyl . The heme a is the accepts an electrons from the copper centre A (two atoms Cu A ). Its function is inhibited by carbon monoxide, CN – , HS – , and N 3 – anions. Heme a of cytochrome aa 3 Heme of cytochrome c

27. Redox p airs in the respiratory chain -0,32 0,00 0,10 0,22 0,24 0,39 0,82 NAD + / NADH+H + FAD / FADH 2 Ubi qu inon e (Q) / Ubi qui nol (QH 2 ) Cytochrom e c 1 (Fe 3+ / Fe 2+ ) Cytochrom e c (Fe 3+ / Fe 2+ ) Cytochrom e a 3 (Fe 3+ / Fe 2+ ) O 2 / 2 H 2 O E  ´ (V) Oxid ized / Redu ced form

29. Entry points for redu cing e qui valent s in R.Ch. I. acyl-CoA enoyl-CoA succinate fumarate cytoplasm shuttle beta-oxidation pyruvate, CAC, KB CAC

30. Enzym e c omplex es in respiratory chain * also called NADH dehydrogenase ! O 2  H 2 O cyt c red  cyt c ox cyt a , a 3 , Cu cytochrom e - c -oxidas e IV. cyt c ox  cyt c red Q H 2  Q Fe-S, cyt b , c 1 Q- cytochrom e- c -redu c tas e III. Q  Q H 2 FADH 2  FAD FAD,Fe-S ,cyt b s u ccinate-Q reductase II. Q  Q H 2 NADH  NAD + FMN, Fe-S NADH- Q oxidoreductase* I. Reduction Oxidation Cofactors Name No.

31. NADH+ H + + Q + 4 H + matrix  NAD + + QH 2 + 4 H + ims Complex I oxidizes NADH and reduces ubiquinone (Q) FMN, Fe-S The four H + are translocated from matrix to intermembrane space (ims)

32. C omplex I oxidizes NADH and translocates 4 H + into intermembrane space VMM 2 H + I. 2 H + 2 H + intermembrane space IMM

33. C omplex II (independent entry) oxidizes FADH 2 from citrate cycle and reduces ubiquinone matrix VMM II. succinate fumarate IMM Complex II, in contrast to complex I, does not transport H + across IMM. Consequently, less proton gradient (and less ATP) is formed from the oxidation of FADH 2 than from NADH.

34. C omplex III oxidizes Q H 2 , reduces cytochrome c , and translocates 4 H + across IMM matrix VMM mezimembránový prostor 2 H + 2 H + III. 4 H + IMM intermembrane space Q-cycle

35. C omplex IV oxidizes cyt c red and two electrons reduce monooxygen ( ½O 2 ) VMM ? IV. 2 H + cyt c red IMM intermembrane space

36. Complex IV: real process is four -electrone reduction of dioxygen complete reaction: 4 cyt-Fe 2+ + O 2 + 8 H + matrix  4 cyt-Fe 3+ + 2 H 2 O + 4 H + ims partial reaction (redox pair) : O 2 + 4 e - + 4 H +  2 H 2 O For every 2 electrons, 2 H + are pumped into intermembrane space

37. Three times translocated protons create electrochemical H + gradient across IMM It consists of two components: 1) difference in pH , Δ G = RT ln ([H + ] out /[H + ] in ) = 2.3 RT(pH out – pH in ) 2) difference in electric potential (Δ  , negative inside), depends not only on protons, but also on concentrations of other ions, Δ G = – n F Δ  The proton motive force Δ p is the quantity expressed in the term of potential (milivolts per mole of H + transferred): Δ p = – Δ G / n F = Δ  + 60 Δ pH .

39. Aerobic phosphorylation of ADP by ATP synthase The phosphorylation of ADP is driven by the flux of protons back into the matrix along the electrochemical gradient through ATP synthase. H + H + H + H + – – – + + + ADP + P i ATP NAD H + H + O 2 2H 2 O NAD + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + H + e – Terminal respiratory chain Electrochemical gradient M-side (negative) C-side (positive)

40. ATP synthase consists of three parts 1) F 1 complex projects into the matrix, 5 subunit types (  3 ,  3 ,  ,  ,  ) catalyze the ATP synthesis 2) connecting section 3) F o inner membrane component, several c -units form a rotating proton channel F 1 head F O segment connecting section

41. ATP -synthase is a molecular rotating motor: 3 ATP/turn H + H + H + H + H + H + H + H + H + F o rotates F 1 does not rotate ADP + P i  ATP a,b, δ subunits hinder rotation of F 1 a α β

43. Control of the oxidative phosphorylation Production of ATP is strictly coordinated so that ATP is never produced more rapidly than necessary. Synthesis of ATP depends on: – supply of substrates (mainly NADH+H + ) – supply of dioxygen – the energy output of the cell; hydrolysis of ATP increases the concentration of ADP in the matrix, which activates ATP production. This mechanism is called respiratory control.

44. Inhibitors of the terminal respiratory chain Complex I is blocked by an insecticide rotenone . A limited synthesis of ATP exists due to electrons donated to ubiquinone through complex II. Complex III is inhibited by antimycin A – complexes I and II become reduced, complexes III and IV remain oxidized. Ascorbate restores respiration, because it reduces cyt c . Complex IV is blocked by carbon monoxide , cyanide ion, HS – (sulfane intoxication), azide ion N 3 – . Respiration is disabled. ascorbate CN – , CO HS – , N 3 – rotenone amobarbital antimycin A

45. occurs after ingestion of alkali cyanides or inhalation of hydrogen cyanide. Bitter almonds or apricot kernels contain amygdalin, which can release HCN. Cyanide ion, besides inhibition of cytochrome c oxidase, binds with high affinity onto methemoglobin (Fe 3+ ). The lethal dose LD 50 of alkali cyanide is about 250 mg. Symptoms - dizziness, gasping for breath, cramps, and unconsciousness follow rapidly. Antidotes may be effective, when applied without any delay: Hydroxycobalamin (a semisynthetic compound) exhibits high affinity to CN – ions, binds them in the form of harmless cyano c obalamin (B 12 ). Sodium nitrite NaNO 2 or amyl nitrite oxidize hemoglobin (Fe II ) to methemoglobin (Fe III ), which is not able to transport oxygen, but binds CN – and may so prevent inhibition of cytochrome c oxidase. Sodium thiosulfate Na 2 S 2 O 3 , administered intravenously, can convert cyanide to the relatively harmless thiocyanate ion: CN – + S 2 O 3 2–  SCN – + SO 3 2– . Cyanide poisoning Carbon monoxide poisoning CO binds primarily to hemoglobin (Fe II ) and inhibits oxygen transport, but it also blocks the respiratory chain by inhibiting cytochrome oxidase (complex IV). Oxygenotherapy improve s blood oxygen transport, administered methylene blue serves as acceptor of electrons from complex III so that limited ATP synthesis can continue.

46. Uncoupling of the respiratory chain and phosphorylation is the wasteful oxidation of substrates without concomitant ATP synthesis: protons are pumped across the membrane, but they re-enter the matrix using some other way than that represented by ATP synthase. The free energy derived from oxidation of substrates appears as heat.. DNP is very toxic, the lethal dose is about 1 g. More than 80 years ago, the long-term application of small doses (2.5 mg/kg) was recommended as a " reliable“ drug in patients seeking to lose weight. Its use has been banned, because hyperthermia and toxic side effect (with fatal results) were excessive. 2 Ionophors that do not disturb the chemical potential of protons, but diminish the electric potential Δ  by enabling free re-entry of K + (e.g. valinomycin) or both K + and Na + (e.g. gramicidin A). 3 Inhibitors of ATP synthase – oligomycin. 4 Inhibitors of ATP/ADP translocase like unusual plant and mould toxins bongkrekic acid (irreversibly binds ADP onto the translocase) and atractylate (inhibits binding of ATP to the translocase). ATP synthase then lacks its substrate. There are four types of artificial or natural uncouplers: 1 " True“ uncouplers – compounds that transfer protons through the membrane. A typical uncoupler is 2,4-dinitropheno l (DNP):

48. The outer membrane is quite permeable for small molecules and ions – it contains many copies of mitochondrial porin (voltage-dependent anion channel, VDAC). The inner membrane is intrinsically impermeable to nearly all ions and polar molecules, but there are many specific transporters which shuttles metabolites (e.g. pyruvate, malate, citrate, ATP) and protons (terminal respiratory chain and ATP synthase) across the membrane. Mitochondrial metabolite transport (the C side and the opposite M side)

49. Transport through the inner mitochondrial membrane Primary active H + transport forms the proton motive force (the primary gradient ) Free diffusion of O 2 , CO 2 , H 2 O, NH 3 cytosolic side positive matrix side negative Ca 2+ pyruvate OH – ADP 3– ATP 4– OH – malate succinate citrate isocitrate aspartate H 2 PO 4 – HPO 4 2– malate malate Secondary active transports driven by a H + gradient and dissipating it: ATP/ADP translocase pyruvate transporter phosphate permease – forms a ( secondary) phosphate gradient dicarboxylate carrier tricarboxylate carrier the malate shuttle for NADH + H +

50. Reactive oxygen species (ROS)

51. Rea ctive oxygen species in human body * Typically phospholipid -PUFA derivatives during lipoperoxidation : PUFA-OO · , PUFA-OOH Neutr al / A nion / C ation Radi cals Hydrogen p eroxid e H-O-O-H Hydrop eroxid e* R-O-O-H Hypochlorous acid HClO Singlet oxygen 1 O 2 Peroxynitrit e ONOO - Nitronium NO 2 + Superoxid e · O 2 - Hydroxyl radi ca l · OH Peroxyl radi cal * ROO · Alkoxyl radi cal RO · Hydroperoxyl radi cal HOO · Nitric oxide NO ·

54. Radi cal •OH is the most reactive species; it is formed from su peroxid e and hydrogen peroxide • O 2 - + H 2 O 2  O 2 + OH - + •OH Catalyzed by redu ced metal ions (Fe 2+ , Cu + ) (Fenton reaction )

56. Hydrogen p eroxid e H 2 O 2 is a side product in the deamina tion of certain amino acids H 2 O + ½ O 2 imino acid catalase oxo acid two-electron reduction

58. Compare: reduction of dioxygen ! O 2 + 2 e - + 2 H +  H 2 O 2 Two-electron O 2 + e -  · O 2 - One-electron O 2 + 4 e - + 4 H +  2 H 2 O Four-electron R edox pair Type of reduction

61. Compounds releasing NO Na 2 [Fe(CN) 5 NO] sodium nitroprus s id e ( natrii nitroprussias ) sodium penta c yanonitrosyl ferrate(III) glycerol trinitr ate ( glyceroli trinitras ) isosorbid dinitr ate ( isosorbidi dinitras ) amyl nitrit e isobutyl nitrit e

63. Bad effects of ROS muta tions t ransla tions errors i nhibi tion of proteosynt hesis d eoxyribos e decomposition m odifi cation of bases chain breaks DNA changes in ion transport influx of Ca 2+ into cytosol altered enzym e activity a g g rega tion, cross-linkage f ragmenta tion oxidation of – SH , ph enyl Protein s changes in membr ane permeability, damage of membr ane enzym es formation of aldehyd es (MDA) and peroxides PUFA Consequences Damage Substrate

67. Lipophilic antioxidants see Harper, Chapter 44 Plant oils, nuts, seeds, germs Fruits, vegetables (most effective is lycopene) Formed in the body from tyrosine Tocopherol Carotenoids Ubiquinol Sources Antioxidant

68. Hydrophilic antioxidants Fruits, vegetables, potatoes Fruits, vegetables, tea, wine Made in the body from cysteine Catabolite of purine bases Made in the body from cysteine L-as corbate Flavonoid s Dihydrolipo ate Uric acid Glutathion e Sources Antioxidant

71. Lycopene does not have the β -ionone ring lycopene beta-carotene

76. L-As c orb ic acid is a weak diprotic acid Two c onjug ate pairs : As corbic acid / hydrogen as c orb ate Hydrogen as c orb ate / as corbate two enol hydroxyl s p K A1 = 4 . 2 p K A2 = 11 . 6

77. L-As c orb ic acid has reducing properties (antioxidant ) as c orb ic acid dehydroas c orb ic acid (redu ced form) (oxid ized form)

84. Uric acid (lactim) is a weak diprotic acid uric acid hydrogen ur ate ur ate p K A1 = 5 . 4 p K A2 = 10 . 3 2,6,8-trihydroxypurin e

85. Uric acid is the most abundant plasma antioxidant stable radi cal (oxid ized form) R · is · OH, superoxid e h ydrogen ur ate (redu ced form) Compare plasma concentrations Ascorbic acid: 10 - 100 μ mol/l Uric acid: 200 - 420 μ mol/l