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Biological Oxidation 生 物 氧 化 Jiao Li Department of biochemistry and molecular biology 021-65986142 [email protected] exercise * How food provide energy? glycogen glucose triglyceride protein Fatty acid+glycerol Amino acid Acetyl-CoA TAC CO2 2H ADP+Pi ATP Respiratory chain Reducing equavalents( H or e-) H2O Learning Objectives After completion of this section, you should be able to : 1. List the components of four protein complexes Ⅰ,Ⅱ,Ⅲ and Ⅳ that involved in electron transport. 2. Describe the electron transport along the protein complexes of respiratory chain. 3. Understand the mechanism of ATP synthesis by oxidative phosphorylation. 4. Illustrate the site of action of common poisons that block electron transport and oxidative phosphorylation. Outline of the section 1. Large four protein complexes Ⅰ,Ⅱ,Ⅲ and Ⅳ embedded in the inner mitochondrial membrane. 2. Electron transport via the respiratory chain. 3. ATP synthesis by oxidative phosphorylation. 4. common poisons that block electron transport and oxidative phosphorylation. 5. Substrates shuttle into mitochondial respiratory chain. Section 1 Large four protein complexes Ⅰ,Ⅱ,Ⅲ and Ⅳ embedded in the inner mitochondrial membrane. glycogen glucose triglyceride protein Fatty acid+glycerol Amino acid Acetyl-CoA mitochondrion TAC CO2 2H ADP+Pi ATP Respiratory chain H2O Mitochondrion The inner membrane is protein rich (4:1 protein:lipid). not permeable and contains within it the electron transporter, ATP synthase, membrane transport proteins. The outer membrane is readily permeable to molecules with molecular weights below 10,000 Da. . ADP+Pi ATP 2H Respiratory chain O2 H2O Transfer of reducing equavalents to oxygen occurs in inner mitochondrial membrane 1. Respiratory chain Definition H+ and e- are passed through a set of electron transporters embedded in inner mt membrane to O2. Respiratory chain comprises enzymatic series electron transporters (electron donors and acceptors), also called electron transfer chain(ETC). Composition Electron transporters(2H 2H+ + 2e) 4 protein complexes + 2 free electron transporter(CoQ and Cyt c ) Human ETC protein complex mw polypeptides cofactor ComplexⅠ FMN, Fe-S (NADH-Q reductase) 880 44 ComplexⅡ FAD, Fe-S, Cytb (succinate-Q reductase) 140 4 ComplexⅢ S, CytbL/H, (Q-Cytc reductase) 280 ≥11 ComplexⅣ Rieske FeCytc1 200 14 CuA、Cyta、 Location of each complex of respiratory chain Cytochrome c cytosol Inner membrane matrix CoQ and Cytochrome c are mobile component. 2. ComplexⅠ (NADH-CoQ reductase) composition: flavoprotein(FMN)、iron-sulphur protein(Fe-S) function: electron tansfer from NADH to CoQ(ubiquinone) NADH NADH + H+ NAD+ FMN; Fe-S FMN FMNH2 CoQ Fe2+S Fe3+S CoQ CoQH2 Structure of NAD+ and NADP+ Nicotinamide R=H: NAD+; R=H2PO3: NADP+ Conversion between NAD+(NADP+) and NADH+H+ (NADPH+H+) or or FMN(flavin mononucleotide) isoallo xazine Fe-S prosthetic group in iron-sulphur protein carries the reaction: Fe2+ Fe3++e- Ⓢ indicates inorganic sulphur Fe-S S Inorganic sulphur S S-cys Ubiquinone (Coenzyme Q, CoQ, Q)contains a long isoprenoid units tail(human:CoQ10) and transfer H+ and e- by conversion as follows. Quinone (oxidized form) Semiquinone (free radical form) Quinone (reduced form) NADH+H+ NAD+ FMN Reduced Fe2+-S Q FMNH2 Oxidized Fe3+-S QH2 Function of complexⅠ matrix cytosol 3. Complex Ⅱ: Succinate-CoQ Reductase composition: flavoprotein(FAD)、iron-sulphur protein(Fe-S)、cytochrome b function: electron tansfer from succinate to CoQ(ubiquinone) succinate→ Complex Ⅱ FAD; Fe-S; b; Fe-S → CoQ isoalloxazine AMP FAD(flavin adenine dinulceotide) Cytochrome A hemeprotein contains heme group and is primarily responsible for transferring electrons. It is divided into several types via their absorbance of wavelength, such as cytochrome a, b, c, etc. protein b a c Function of complex Ⅱ cytosol matrix succinate Succinate Fumarate FAD FADH2 Fe2+S Fe3+S CoQ CoQH2 4. Complex Ⅲ: CoQ-Cytochrome c Reductase composition: cytochrome bL/bH, iron-sulphur protein(Rieske Fe-S ), cytochrome c1 function: electron tansfer from CoQ(ubiquinone) to cytochrome c • CoQ passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q cycle. Complex Ⅲ QH2→ b; Fe-S; c1 →Cyt c One e- is transferred to cyt c Second e- is back to Q Q cycle in complex Ⅲ catalyzed electron transfer 5. Complex Ⅳ: Cytochrome c Oxidase composition: CuA, cytochrome a/a3, CuB function: electron tansfer from cytochrome c to O2. Cyt a3 /CuB binuclear center transfers e- to O2。 Complex Ⅳ Reduced Cyt c → CuA→a→a3→CuB → O2 • Oxygen is thus the terminal acceptor of electrons in the electron transport pathway - the end! cytosol matrix Function of Complex Ⅳ Section 2 Electron transport via the respiratory chain 1. Direction of electron flow in ETC is from experiment Experiments included: ① determination of standard redox potential ② separation and recombination of each component. ③ specific inhibitors ④ oxygen is added to reduced ETC at a slow rate. Standard reduction potentials of the major respiratory electron carriers. • Electrons generally fall in energy through the chain - from complexes I and II to complex IV 2. Reducing equavalence enter ETC by two routes 1. NADH redox respiratory chain NADH →complexⅠ→Q →complex Ⅲ→Cyt c →complex Ⅳ→O2 2. Succinate redox respiratory chain Succinate →complexⅡ →Q →complex Ⅲ→Cyt c →complexⅣ→O2 NADH respiratory chain FADH2 respiratory chain complex Ⅱ complex Ⅰ complex Ⅲ complex Ⅳ Electron transport occurs in inner mitochondrial membrane via a series of protein complexes. ※ complex Ⅰ,Ⅲ, Ⅳ are also a pump to pump out H+ to intermembrane space. Section 3 ATP synthesis by oxidative phosphorylation 1. Oxidative phosporylation provides most energy captured during catablism * Definition Electron transfer from reducing equavalents to oxygen via ETC couples the synthesis of ATP from ADP, which is oxidative phosphorylation, also called coupled phosphorylation. Substrate level phosphorylation another way to synthesize ATP. is 2. Coupling sites of oxidative phosphorylation in ETC Experiments: (1) P:O ratio (2) Free energy change- ΔG°′ (1) P:O ratio:For per half mol O2, the number of inorganic phosphate consumed is equal to the number of ATP generated. 线粒体离体实验测得的一些底物的P/O比值 P:O ratio determined from mitochondrion in vitro substrate 底 物 β-hydroxybutyric β-羟丁酸 acid Respiratory 呼吸链的组成chain NAD+→复合体Ⅰ→CoQ→复合体Ⅲ complexⅠ complexⅢ P:O ratio P/O比值 2.4~2.8 Number of ATP 可能生成的 ATP数 3 complexⅣ →Cyt c→复合体Ⅳ→O 2 琥珀酸 Succinate complexⅢ 复合体Ⅱ→CoQ→复合体Ⅲ complexⅡ 1.7 2 complexⅣ →Cyt c→复合体Ⅳ→O 2 抗坏血酸 Ascorbic acid Cytochrome c 细胞色素c complexⅣ Cyt c→复合体Ⅳ→O 2 (Fe2+) complexⅣ 复合体Ⅳ→O 2 0.88 1 0.61-0.68 1 (2) Redox potential in respiratory chain 呼吸链中各种氧化还原对的标准氧化还原电位 氧化还原对 System complex Ⅰ -70.4kJ/mol complex Ⅲ -36.7kJ/mol complex Ⅳ -83kJ/mol NAD+/NADH+H+ FMN/ FMNH2 FAD/ FADH2 Cyt b Fe3+/Fe2+ Q10/Q10H2 Cyt c1 Fe3+/ Fe2+ Cyt c Fe3+/Fe2+ Cyt a Fe3+ / Fe2+ Cyt a3 Fe3+ / Fe2+ 1/2 O2/ H2O ΔG°′= - nFΔ E°′ Eº' (V) -0.32 -0.30 ATP → ADP + P i ΔG°′= −30.5kJ/mol -0.06 0.08 0.04(或0.10) 0.07 0.22 0.25 0.29 0.55 0.82 ΔG°′: free energy change Δ E°′: potential change n: number of electrons F : constant(96.5 kJ /mol · V ) Coupling sites:ComplexⅠ, Ⅲ, Ⅳ Succinate Complex Ⅱ Coupling sites of oxidative phosphorylation ATP ATP ComplexⅠ Complex Ⅲ ATP ComplexⅣ How ATP is produced ? 3. Chemiosmotic theory account for oxidative phosphorylation. ComplexⅠ, Ⅲ, Ⅳ act as a proton pump. H+ in electron flow is pumped out to cytosol to form electrochemical gradient across inner membrane. Back flow of H+ drives generation of ATP from ADP and Pi. ComplexⅠ, Ⅲ, Ⅳ act as a proton pump Cytosol 4H+ + Cyt c 4H+ + + + + + 2H+ + 4H+ + + Q - Ⅰ F Ⅱ - - Ⅲ NAD+ Succinate 0 Ⅳ - - - Fumaric acid NADH+H+ + 1/2O2+2H+ - - - H2O F1 Matrix ADP+Pi ATP H+ 4. ATP synthase (ComplexⅤ) Cytosol Inner membrane Matrix ATP synthase model 4. ATP synthase (complexⅤ) b F1:hydrophilic, ( α3β3γδε subunits ) F0:hydrophobic, (a1b2c9~12 subunits) Fo not F0 since O stands for oligomycin! c a Structure: F0 δ γ ε F6 Catalytic subunit β OSCP Catalytic subunit β α ATP synthase F1 Mechanism of ATP production by ATP synthase. Pro-tons passing through the disk of “C” units cause it and γ-subunitRed: to rotate. OADP and P i are taken up sequentially byOrange: the β-subunitsLto form ATP, which is expelled as Pink: the rotating T γ-subunit squeezes each β-subunit in turn. Thus, three ATP molecules are generated per revolution. H+ flow 5. Factors affecting oxidative phosphorylation ADP (major regulation) Rate of respiratory in Mt is mainly controlled by availability of ADP ADP O2 consumption Resting state 6. ATP (1) High energy bond and high energy phosphate compound High energy phosphate bond ≥21kJ/mol free energy is release during hydrolysis of some phosphate ester bond or phosphate anhydride bond, P. High energy phosphate Standard free energy of hydrolysis of some organophosphates of biochemical importance γ β ~ ~ α (2) Synthesis and use of ATP ATP creatine Creatine phosphate oxidative phosphorylation ~P substrate phosphorylation ADP ATP is the center of energy generation and use ~P mechanic(contraction) osmotic(active transport) chemical(anabolism) electrical(bioelectricity) heat(body temperature) Section 4 Common poisons that block electron transport and oxidative phosphorylation 1. Many poisons inhibit respiratory chain (1) Electron transport inhibitor Piericidin A Amobarbital Rotenone (2) Oxidative phosphorylation inhibitor Oligomycin (3) Oxidative phosphorylation uncoupler 2,4-dinitrophenol Thermogenin (physical uncoupler) Site of action of some poisons 2. Mechanism of action of uncoupler H NO2 NO2 DNP: 2,4-dinitrophenol O2N OH O 2N O H+ Cytosol Cyt c uncoupler Q Ⅰ Ⅱ F Ⅲ Ⅳ Matrix 0 F1 ADP+Pi ATP H+ Thermogenin (physical uncoupler) provide heat for body. Heat 3. Mechanism of action of oligomycin cytosol Oligomycin blocks H+ flow through c channel of ATP synthase matrix oligomycin ATP synthase Section 5 Substrates shuttle into mitochondrial respiratory chain 1. Transporter in inner mitochondrial membrane Impermeability of inner membrane requires transporter to help transport substrate and substance. 2. Shuttle systems Cytoplasmic NADH enter into mitochondrial matrix through two shuttles. Shuttle 1: α-glycerophosphate shuttle Shuttle 2: malate-asparate shuttle (1) α-glycerophosphate shuttle (brain and white muscle) CH2OH CH2OH ETC NADH+H+ α-glycerolphosphate dehydrogenase NAD+ C=O C=O CH2O- Pi CH2O- Pi Dihydroxyacetone phosphate αglycerolphosphate dehydrogenase CH2OH CH2OH CHOH CHOH CH2O- Pi CH2O- Pi α-glycerolphosphate FADH2 FAD inner outer Intermembrane membrane membrane space matrix (2) Malate-asparate shuttle (universal) (3) Adenine nucleotide transporter ADP3- ATP4- 3H+ H2PO4- H+ 胞液侧 F 0 基质侧 腺苷酸 转运蛋白 F1 ATP4- 磷酸 转运蛋白 H2PO4- H+ ADP3H+ ATP and ADP are antiport in inner membrane Clinical case MELAS ( Mitochondrial encephalopathy, lactic acidosis, and stroke ) 1. Genetic disease due to mutations in genes of mitochondrial DNA( eg. NADH-Q reductase) 2. Brain and muscle are mainly affected. Summary 1. Four protein complexes Ⅰ,Ⅱ,Ⅲ and Ⅳ are mainly responsible for transferring electron in ETC. 2. ATP is synthesized by oxidative phosphorylation driven by H+ gradient . 4. Some poisons inhibit biologic oxidation by blocking electron transport and oxidative phosphorylation. Study question 1. Which of following subunit is mainly responsible to catalyze the synthesis of ATP in ATP synthase? A. α B. β C. γ D. ε E. c 2. What is the consequence of thermalgenin effect. A. Producing more heat B. ETC is inhibited C. Producing more ATP D. Oxidative phosphorylation is increased