Chapter 6 Biological Oxidation PDF

COMPANY LOGO Chapter 6 Biological Oxidation  Biological oxidation is a cellular process in which the organic substances release energy, produce CO2 and H2O through oxidative reductive reactive reactions. lnmuyangjing@126....

COMPANY LOGO Chapter 6 Biological Oxidation  Biological oxidation is a cellular process in which the organic substances release energy, produce CO2 and H2O through oxidative reductive reactive reactions. [email protected] General introduction of oxidation Oxidative-reductive reactions Nutrients + O2 H2O + CO2 + ATP [email protected] Forms of oxido-reduction in biological oxidation Oxidative reaction* Loss of electrons Removal of hydrogen Addition of oxygen Reductive reaction Gain of electrons Addition of hydrogen Removal of oxygenation [email protected]  Oxidation in vivo and in vitro Biological Chemical oxidation oxidation Temperature 37℃ high Condition pH7, liquid dry Catalyst Enzyme Form of energy ATP heat Producing CO2 organic acid decarboxylation Oxygen directly produce CO2; wide addition of combine with and H2O carbon(hydrogen) water and dehydrogenation , the hydrogen combine with , produce oxygen in indirect way. CO2(H2O).  Enzymes involved in oxidation and reduction are called oxidoreductase. Classified into four group: Oxidases Dehydrogenase Hydroperoxidases oxygenase [email protected]  Oxidases use oxygen as a hydrogen acceptor Oxidases do not incorporate oxygen into the substances in the oxidation reactions they catalyze. these enzyme catalyze the removal of hydrogen from a substrate using product. AH2 AH2 ½ O2 O2 Red Red Oxidases Oxidases A A H2O H2O2 Ox Ox [email protected]  Dehydrogenases use coenzymes as hydrogen acceptor In the reaction catalyzed by dehydrogenase, the oxidation of one substrate is accompanied by the reduction of another substrate so that the reaction occurs in the absence of oxygen. [email protected] AH2 Carrier BH2 (Red) (OX) (Red) A Carrier-H2 B (Ox) (OX) (Red) Dehydrogenase Dehydrogenase Specific for A Specific for B [email protected] [email protected]  COMPANY LOGO 6.1 Electron Transport Chain Mitochondria [email protected] Mitochondria inner membrane [email protected]  Components of respiratory chain Complex Name Number of Prosthetic group peptide chain ComplexⅠ NADH-CoQ 42 FMN, Fe-S reductase ComplexⅡ Succinate-CoQ 4 FAD, Fe-S reductase ComplexⅢ CoQ-Cyt C 11 Fe-S, iron reductase protoporphyrin, ComplexⅣ Cyt C oxidase 13 Cu, iron protoporphyrin  CoQ and Cyt C are soluble and more mobile [email protected]  (1) ComplexⅠ: NADH-CoQ oxidoreductase; or NADH dehydrogenase Intermembrane space Composition: matrix iron-sulfur protein (iron-sulfur cluster) Flavoprotein (FMN) Functions: Bind and oxidize NADH, transfer electrons to CoQ, release 4H+ to interspace between inner and outer membrane. [email protected]  R=H: NAD+; R=H2PO3: NADP+ NAD+ ——nicotinamide adenine dinucleotide NADP+ ——nicotinamide adenine dinucleotide phosphate [email protected] Transformation of NAD+ (NADP+) and NADH (NADPH) quinquevalent nitrogen trivalent nitrogen NAD+ (NADP+) NADH (NADPH) [email protected]  Transformation of FMN and FMNH2 FMN contains riboflavin, and the functional group is iso- alloxazine. The two nitrogen atoms can receive and release hydrogen. Iso-alloxazine [email protected]  Iron-sulfur protein Fe2+ Fe3+ + e Fe-S cluster Fe-S 2Fe-2S 4Fe-4S [email protected]  Ubiquinone,Q (Coenzyme Q,CoQ)  quinones contain a polyisoprene side chain.  liposolubility ， moving in mitochondrial inner membrane easily.  the only one electron carrier without protein in respiratory chain.  Ubiquinone( oxidized) Semiquinone (QH˙) Ubiquinol (QH2, reduced) Transformation of Q and QH2 [email protected] Complex I ： NADH→ FMN→ Fe-S→ CoQ→ Fe-S→ CoQ [email protected]  (2) Complex II: Succinate-CoQ reductase Succinate dehydrogenase complex Intermembrane space matrix succinate Functions: Transfer electron from succinate to Q, do not release H+ to the interspace. [email protected]  (3) Complex III: CoQ-Cyt c reductase Composition: iron-sulfur protein (iron-sulfur cluster) hemeprotein—— Cytochrome (heme) Cyt b562, Cyt b566 Cyt c1 Functions: electron transfer from CoQ to Cyt c. Every two electrons transferring lead to 4H+ pumped to the inter space. [email protected]  Cytochrome (Cyt) A. Structure: colorant protein containing iron porphyrin. B. Typing: Cyt a: Cyt aa3 Cyt b: Cyt b562 、 Cytb566 、 Cytb560 Cyt c: Cyt c 、 Cyt c1 C. Difference: different side chain of iron porphyrin. different linkage form of iron porphyrin with the protein. Cyt Fe3+ + e  CytFe2+ Cyt c [email protected]  polyisoprene chain ethenyl Formyl group [email protected]  Difference between Cyt a and Cyt b, Cyt c band prothetic group color wavelength linkage with protein Non-covalent Cyta heme a green 600nm bonding Non-covalent Cytb heme b red 560nm bonding Bind with-SH of Cytc heme c red 550nm Cys [email protected]  (4) Complex IV: Cytc oxidase Composition: Copper-containing protein (Cu2+ ) Cu2+ + e  Cu+ Hemeprotein——Cytochrome (heme) Cyt a, Cyt a3 Functions: electron transfer from Cyt c to H2O. Every two electrons transferring lead to 2H+ pumped to the inter space. [email protected] ComplexⅠ is responsible for transferring electrons from NADH to CoQ. Complex Ⅱ is responsible for transferring electrons from succinate to the physiological acceptor CoQ. Complex Ⅲ is responsible for transferring electrons from QH2 to Cytc. Complex Ⅳ is responsible for transferring electrons from cytochrome c to oxygen. [email protected]  Positions of the complexes in respiratory chain ① Standard redox potential of the component (E0’) ② Absorption spectrums of the component in oxidation and reduction state respectively ③ Specific blocking agents ④ Reconstruction of respiratory chain in vitro [email protected] Standard redox potentials of respiratory chain and related electron carriers [email protected]  A chain in the mitochondria consists of a number of redox carriers for transferring hydrogens removed from the substrate to oxygen to form water. The chain is termed a respiratory chain*, also called electron transport chain (ETC)*. [email protected] It is an oxido-reduction system which consists of a series of enzymes, coenzyme aligning in mitochondrial inner membrane, transfer hydrogen and electron from the substrate to oxygen to form water and produce ATP. [email protected] Sites in respiratory chain producing energy for ADP phosphorylation Free energy change in respiratory chain potential change Free-energy change ATP producing ? sites (ΔE0’) ΔG0’=nFΔE0’ ΔG0’>30.5 KJ ? Yes Yes Yes [email protected]  succinate succinate -hydroxybutyrate FAD Vit C £¨ Fe-S£© NADH FMN CoQ Cyt b Cyt c1 Cyt c Cyt aa3 O2 £¨ Fe-S£© E0' -0.32 -0.22 +0.04 +0.08 +0.23 +0.25 +0.29 +0.82 ∆E0' 0.36V 0.21V 0.53V ∆G0' 69.5kJ/mol 40.5kJ/mol 102.3kJ/mol energy energy energy ADP + Pi ATP ADP + Pi ATP ADP + Pi ATP [email protected] There are two respiratory chains* 1.NADH-linked respiratory chain NADH →complex Ⅰ→Q → complex Ⅲ →Cyt c →complex Ⅳ→O2 2. Succinate-linked respiratory chain Succinate → complex Ⅱ →Q → complex Ⅲ →Cyt c → complex Ⅳ→O2 [email protected]  Cytc Intermembrane e- space e- Q e- e- Inner membrane Ⅱ e- Ⅳ Ⅰ Ⅲ NADH+H+ fumarate H 2O matrix 1/2O2+2H + NAD + succinate [email protected]  The respiratory chain provides most of the energy captured in metabolism When substrate are oxidized via an NAD+-linked dehydrogenase and the respiratory chain, approximately 2.5 molecules of ATP are synthesized for each pair of electrons transferred from NADH to O2 in the NADH respiratory chain. [email protected]  On the other hand, when a substrate is oxidized via a flavoprotein-linked dehydrogenase, approximately 1.5 molecules of ATP are formed. These reactions are known as oxidative phosphorylation at the respiratory chain level. [email protected] [email protected] 6.2 Oxidative phosphorylation*: The process of the phosphorylation of ADP to produce ATP is coupled with the process of electron transfer in the respiratory chain in mitochondria. [email protected]  NAD+-----------NADH FAD-------------FADH2 As was the case for carbohydrates and lipids, the degradation of amino acids results ultimately in the generation of reducing equivalents (NADH and FADH2). [email protected]  Chemiosmotic theory What’s the way of ADP phosphorylation coupled with respiratory chain oxidation ？ the energy of respiratory chain oxidation change into proton gradient across the inner membrane. the proton gradient drive ATP- synthase produce ATP. Proposed in 1961, Nobel prize in 1978 [email protected] Nobel prize in 1997 [email protected]  Conceptual diagram of chemiosmotic theory 2H+ Intermembrane 4H + 4H+ space fumarate succinate matrix H+ concentration difference The energy of the H+ gradient drives ATP synthesis [email protected] ATP synthase [email protected]  ATP synthase  Consists of hydrophobic F0(a1b2c912) and hydrophilic F1(33).  When proton go straight through α, push c ring anticlockwise turning , and as a result, spur the γ turning.  The rotation of γ change the conformation of β, which causes the conversion of ADP+Pi into ATP. [email protected] [email protected] [email protected] Generate ATP sums 2.5 ATP are synthesized per NADH oxidized through the NADH respiratory chain 1.5 ATP are synthesized per FADH2 oxidized through the FADH2 respiratory chain [email protected] P/O ratio* Number of moles of ATP produced as consuming a mole of oxygen atom in a reaction, i.e. the number of moles of phosphor consumed when consuming a mole of oxygen atom in the reaction. P/O ratio determined by experiment: NADH oxidative respiratory chain——P/O=2.5 Succinate oxidative respiratory chain——P/O=1.5 [email protected]  P/O ratio of some substrate Number substrates components P/O ratio of ATP -hydroxybutyrate NAD+→O2 2.4~2.8 3 succinate FAD →O2 1.7 2 Vit C Cyt c→O2 0.88 1 Cyt c Cyt aa3→O2 0.61~0.68 1 [email protected]  Inhibitors of oxidative phosphorylation  Regulation by ADP  Inhibitors  Thyroid hormone  Mitochondrial DNA mutation [email protected] (1) Regulation by ADP main regulation factor ： ADP/ATP ratio ADP/ATP ↑——oxidative phosphorylation ↑ ADP/ATP ↓——oxidative phosphorylation ↓ [email protected] (2) Inhibitors Inhibitors of respiratory chain block electron transfer. Uncoupler destroy the coupling of oxidation with phosphorylation, like uncoupling protein, 2,4- dinitrophenol. Inhibitors of oxidative phosphorylation restrain the proton return to matrix side in ATP synthase, like oligomycin. [email protected] Inhibitors of respiratory chain H 2S succinate Antimycin A CO Dimercaptopropan CN- ol    一氧化碳中毒（ carbonmonoxide poisoning ) colorless, tasteless Rotenone Amobarbital Piericidi A [email protected]  Uncoupling reagents dissolve in the membrane and function as carriers for H+. [email protected]  Distribution of brown adipose tissue in a newborn infant At birth, human infants have brown fat distributed as shown here, to protect the major blood vessels and the internal organs. This brown fat recedes over time, so that an adult has no major reserves of brown adipose. [email protected]  Inhibitors of oxidative phosphorylation Intermembrane space restrain the proton return to matrix side in ATP oligomycin synthase matrix c ring inhibitory action of oligomycin [email protected] (3) Thyroid Hormone Thyroid Hormone Na+-K+ ATP enzyme Expression of uncoupling protein ATP degradation ATP synthesis ADP Oxidative phosphorylation Oxidative phosphorylation [email protected]  (4) Mitchondrial DNA mutation  13 peptides (7 peptides in NADH dehydrogenase, 1 in Cytc reducase, 3 in Cyt Mitochondrial DNA c oxydase, 2 in ATP synase ) coded by mitochondrial DNA join in oxidative phosphorylation.  Naked cyclic double helix DNA lacks of defend system and restoration encodes only for a few of the proteins system. required for mitochondria function, and  Mutations affect oxidative some mitochondrial rRNA and tRNAs phosphorylation and ATP production.  Symptoms are dependent on the degree of mutation and the different organs need for ATP. [email protected] 6.3 ATP and other high energy compounds Ester bond Adenine NH2 N N O O O HO P O P O P O CH2 N N O OH OH OH r β α OH OH ATP Glycosidic bond Ribose Adenosine triphosphate: ATP [email protected] α bond △ G0′ ＝ － 14.3KJ β bond △ G0′ ＝ － 32.2KJ γ bond △ G0′ ＝ － 30.5KJ >30KJ （ 7kcal ） -high energy bonds [email protected] ATP and other energy-rich compounds Classification of stored energy compounds organic phosphate α-glycerol phosphate compounds phosphate (low-energy compounds ) glucose-6-phosphate compounds ΔG0’ 9~16 KJ/mol ATP phosphoanhydride ADP (high-energy compounds) creatine phosphate ΔG0’ 30~60 KJ/mol 1, 3-bisphosphoglycerate Phosphoenol pyruvate thioester (high-energy compounds) acetyl CoA succinyl-CoA acyl CoA [email protected] Examples of energy-rich compounds Energy-rich compounds ΔG0’ （ pH7.0,25℃) ATP 、 UTP 、 CTP 、 GT 30.5 kJ/mol P 1,3-bisphoglycerate 61.9 kJ/mol Phosphoenoyl pyruvate Creatine phosphate 43.9 kJ/mol Acetyl CoA 31.4 kJ/mol Succinyl CoA Acyl CoA [email protected] Adenylate Kinase: 2 ADP  ATP + AMP Nucleoside Diphosphate Kinase catalyzes reversible reactions such as: ATP + GDP  ADP + GTP, ATP + UDP  ADP + UTP, ATP + CDP  ADP + CTP. [email protected]  High-energy phosphate act as the “energy currency” of the cell ATP is therefore the “energy currency” for living systems, because cells usually transfer phosphate by coupling reaction to ATP hydrolysis. [email protected]  Phosphocreatine provides a “high- energy” reservoir for ATP formation Phosphocreatine is used in nerve and muscle for storage of ~P bonds. H NH2 N P C NH creatine C NH kinase H3C N + ATP H3C N + ADP CH2 CH2 COOH COOH creatine creatine phosphate [email protected] synthesis and consumption of ATP The synthesis of ATP from ADP and Pi is accomplished through two types of processes:* Substrate-level phosphorylation Oxidative phosphorylation [email protected]  The methods for producing ATP* Substrate level phosphorylation: is the direct reaction in which high-energy phosphate group in substrate is captured by ADP to form ATP. Oxidative phosphorylation: is the indirect way of generating ATP from ADP and pi using the energy released from redox reactions on the electron transport. [email protected] 1. Substrate level phosphorylation* ： ATP may be formed through direct transfer of a phosphoryl group from a “high- energy” compound to ADP. this process is known as “substrate level phosphorylation” [email protected] There are 3 reactions in which ATP can be generated by substrate-level phosphorylation. Two reactions are in the glycolysis, and the other one is in the citric acid cycle. [email protected] Substrate level phosphorylation 1) 1,3-bisphosphoglycerate ADP Glycolysis Phosphoglycerate kinase ATP 3-phosphoglycerate [email protected]  2) Phosphoenolpyruvate ADP Glycolysis Pyruvate kinase ATP Pyruvate 3) succinyl CoA GDP TAC succinyl CoA synthetase GTP succinate [email protected]  Selective transport across the inner mitochondrial membrane The outer mitochondrial membrane contains porin, a protein that forms nonspecific pores that permit free diffusion of up to 10 kD molecules. [email protected] 6.4 Selective transport across the inner mitochondrial membrane The inner mitochondrial membrane is freely permeable only to O2,CO2, and H2O. [email protected]  The main transport proteins in inner membrane of mitochondria Transport Function protein of Cytosol Matrix of mitochondria α-ketoglutaric acid malate α-ketoglutaric acid acidic amino acid glutamic acid aspartic acid phosphate H2PO4-H+ H2PO4-H+ adenylic acid ADP ATP pyruvic acid pyruvic acid OH- tricarboxylic acid malate citrate basic amino acid ornithine citrulline carnitine fatty acyl carnitine carnitine [email protected]  Two shuttle systems for Oxidation of cytosolic NADH from glycolysis  malate-aspartate shuttle  glycerol 3-phosphate shuttle (α-glycerophosphate shuttle) [email protected]  - Glycerol 3 phosphate shuttle system Brain, skeletal muscle [email protected]  Note: Location: Brain, skeletal muscle. The shuttle does not allow cytoplasm NADH to enter the mitochondrion, but transports the two electrons from NADH into the mitochondria, and feed the electron into the FADH2 electron transport chain. So synthesize 2 ATPs. [email protected]  Malate-aspartate shuttle ( heart , liver ) glutamate – + H 3N + aspartate H 3N - - carrier - - Respiratory OOC-CH2-C-COO OOC-CH2-C-COO H asparate chain O H + + O -OOC-CH2-C-COO- H3N H3N -OOC-CH2-C-COO- oxaloacetate - OOC-CH2-CH2-C-COO - - - OOC-CH2-CH2-C-COO NADH H glutamate H NADH +H+ +H+ inner aspartate memebrane aminotransferase malate O O dehydrogenase -OOC-CH2-CH2-C-COO- -OOC-CH2-CH2-C-COO- NAD+ NAD + α-ketoglutarate OH OH -OOC-CH2-C-COO- -OOC-CH2-C-COO- H malate H α-ketoglutarate malate malate carrier matrix intermembrane space Note This cycle of reactions is to transfer the electrons from NADH in the cytosol to NADH in the mitochondrial matrix, The NADH in the mitochondria is then re-oxidized by the NADH electron transport chain So synthesize 3ATPs [email protected] In summary : Cytosol NADH go into the mitochondria by this two shuttele  glycerol 3-phosphate shuttle in the mitochondria go into the FADH2 respiratory chain. so produce 2 ATPs Malate-asparate shuttle: in the mitochondria go into the NADH respiratory chain. so produce 3ATPs [email protected]  3H+ 3H+ Transport of ATP, ADP, & Pi [email protected] Terms: 1 ． Biological oxidation 2 ． Respiratory chain 3 ． Oxidative phosphorylation 4 ． Substrate level phosphorylation 5 ． P/O ratios Question: List the order of two important respiratory chains [email protected]