Chapter 7 Biological Oxidation

Biological oxidation is the cellular process in which the organic substances release energy (ATP), produce CO2 and H2O through oxidative-reductive reactions. organic substances: carbohydrate, fat and protein

7.1 Principal of Redox Reaction The electron-donating molecule in a oxidation-reduction reaction is called the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant: for example: Fe 2+ (ferrous) lose -e Fe 3+ (ferric) gain +e

Several forms of Biological Oxidation 1. Loss of electrons 2. Dehydrogenation 3. Oxygenation Redox reaction = reduction-oxidation reaction Several forms of Biological Reduction 1. Gain of electrons 2. Hydrogenation 3. Deoxygenation

Áoxidation-reduction potential ( or redox potential), E : it is a measure of the affinity of a substance for electrons. It decide the loss (or the gain) of electrons. ÁA positive E: the substance has a higher affinity for electrons, accept electrons easily. A negative E: the substance has a lower affinity for electrons, donate electrons easily.

 E0`, the standard redox potential for a substance :is measured under stander condition(25 ℃, 1mmol/L reaction substance),at pH7, and is expressed in volts.

Section 7.2 Respiration Chain and Oxidative Phosphorylation

7.2.1 Respiratory Chain Term: A chain in the mitochondria consists of a number of redox carriers for transferring electrons from the substrate to molecular oxygen to form oxygen ion, which combines with protons to form water.

1.Complex I: NADH:ubiquinone oxidoreductase NADH:CoQ oxidoreductase 2.Complex II: Succinate dehydrogenase 3.Complex III: cytochrome bc1 (ubiquinone Cyt c oxidoreductase ) 4.Complex IV: cytochrome oxidase Redox carriers including 4 protein complexes

Complex I ( NADH:ubiquinone oxidoreductase) Function: transfer electrons from NADH to CoQ Components: NADH dehydrogenase (FMN) Iron-sulfur proteins (Fe-S) complex Ⅰ NADH→ →CoQ FMN; Fe-S N-1a,b ; Fe-S N-4 ; Fe-S N-3 ; Fe-S N-2

R=H: NAD + ; R=H 2 PO 3 :NADP + 1. NAD(P) + : Nicotinamide Adenine Dinucleotide Phosphate)

Oxidation of NADH is a 2- electron(2e), 2-proton(2H) reaction NAD + or NADP + NADH or NADPH

2. FMN can transfer 1 or 2 hydride ions each time Accepts 1 H + and 1 e - to form semiquinone = stable free radical Accepts 2 H + and 2 e - to give fully reduced form FMN: flavin mononucleotide

3. Iron-sulfur clusters (Fe-S) transfers 1-electron at a time, without proton involved Fe 3+ +e - Fe 2+

4.Ubiquinone (CoQ) is lipid-soluble, not a component of complex Ⅰ, can transfer 1 or 2 hydride ions each time. Function: transfer electrons and protons from complex Ⅰ, Ⅱ to complex Ⅲ.

NADH+H + NAD + FMN FMNH 2 Reduced Fe-S Oxidized Fe-S Q QH 2 Matrix Intermembrane space

Complex II : Succinate dehydrogenase (Succinate: CoQ oxidoreductase ) Function: transfer electrons from succinate to CoQ Components: Succinate dehydrogenase (FAD, Fe-S) Cytochrome b 560 Complex Ⅱ Succinate→ →CoQ Fe-S 1 ; b 560 ; FAD; Fe-S 2 ; Fe-S 3

Fe3++e- Fe2+ Cytochromes a, b, c are heme proteins, their heme irons participate redox reactions of e- transport.

Succinate Matrix Intermembrane space

Complex III: cytochrome bc1 (ubiquinone Cyt c oxidoreductase) Function: transfer electrons from CoQ to cytochrome c Components: iron-sulfur protein cytochrome b(b 562, b 566 ) cytochrome c 1 complex Ⅲ QH 2 → →Cyt c b 562 ; b 566 ; Fe-S; c 1

Matrix Intermembrane space Cytochrome c is soluble, which will transfer electrons to complex Ⅳ

Complex IV: cytochrome oxidase Function: transfer electrons from Cyt c to molecule oxygen, the final electron acceptor. Components: cytochrome aa 3 copper ion (Cu 2+ ) Cu 2+ + e - Cu + Complex IV Cyt c → → O 2 Cu A →a→a 3 →Cu B

Coenzyme Q ubiquinone/ol Cytochrome c

Sequence of respiratory chain Principles: e - tend to flow from a redox pair with a lower E°to one with a higher E° In the e - -transport chain, e - -carriers are arranged in order of increasing redox potential, making possible the gradual release of energy stored in NADH, FADH 2

Redox potential redox pair E 0

There are two respiratory chains NADH respiratory chain NADH Complex Ⅰ CoQ Complex Ⅲ cytochrome c Complex Ⅳ O 2 Succinate (FADH 2 ) respiratory chain Succinate Complex Ⅱ CoQ Complex Ⅲ cytochrome c Complex Ⅳ O2

NADH respiration chain FADH 2 respiration chain

The oxidation of organic nutritions produces the energy-rich molecules, NADH and FADH 2. The oxidation of NADH or FADH 2 in mitochondrial is the electron transferring through respiration chain. The free energy produced in electron transferring supports the phosphorylation of ADP to form ATP. The oxidation of NADH or FADH 2 and the formation of ATP are coupled process, called Oxidation Phosphorylation Oxidative Phosphorylation

The Chemiosmotic Theory The free energy of electron transport is conserved by pumping protons from the mitochondrial matrix to the intermembrane space so as to create an electrochemical H + gradient across the inner mitochondrial membrane. The electrochemical potential of this gradient is harnessed to synthesize ATP. Peter Mitchell

Electrochemical H + gradient (Proton-motive force) 2 components involved 1. Chemical potential energy due to difference in [H + ] in two regions separated by a membrane 2. Electrical potential energy that results from the separation of charge when a proton moves across the membrane without a electron.

Complex I: 4 H + expelled per e - -pair transferred to Q Complex III: 4 H + expelled per e - -pair transferred to Cyt c Complex IV: 2e H + from matrix convert ½ O 2 to H 2 O; 2 further H + expelled from matrix

Conformation 1 (high affinity for H + ) Conformation 2 (low affinity for H + ). Proton pumping: Reduction- dependent conformational switch of an e--transport complex

Inner Membrane ATP Synthase Intermembrane space Matrix (ab 2 c 9-12 ) (α 3 β 3 γδε) C ring

Each of 3  -subunits contains an active site F1: multisubunit complex that catalyzes ATP synthesis F 0 = proton-conducting transmembrane unit β-subunit take up ADP and Pi to form ATP ADP + Pi ATP

H + flow β-subunit has three conformations:T (tight), L (loose), O (open) When protons flow back through F 0 channel, γ-subunit is rotated by the rotation of c ring, then the conformations of β-subunits are changed, this lead to the synthesis and release of ATP. To form a ATP need 3 protons flow into matrix.

F0F0 F1F1 胞液侧 基质侧 ATP 4- ADP 3- H 2 PO 4 - ATP 4- H + H+H+ H 2 PO 4 - ADP 3- H+H+ H+H+ Intermembrane space Matrix Translocation of ATP, ADP and Pi.

P/O ratios P/O ratio is the rate of phosphate incorporated into ATP to atoms of O 2 utilized. It measure the number of ATP molecules formed per two electrons transfer through the respiratory chain. NADH respiratory chain : 2.5, FADH 2 respiratory chain: 1.5

During two electrons transfer through NADH respiratory chain, ten protons are pumped out of the matrix. To synthesis and translocation an ATP, four protons are needed. So, two electrons transport can result in 2.5 ATP. To succinate respiratory chain, two electrons transport can result in 1.5 ATP.

Regulation of Oxidative Phosphorylation 1.PMF (proton motive force) regulate the electron transport. higher PMF lower rate of transport 2.ADP concentration resting condition: energy demanded is low, ADP concentration is low, the speed of Oxidative Phosphorytion is low. active condition: the speed is high.

Inhibitor of Oxidative Phosphorylation 1.Inhibitor of electron transport × × × Retonone Amytal Antimycin A SuccinateCyanide, Azide Carbon Monoxide

Ⅰ Ⅳ Cyt c F0F0F0F0 F1F1F1F1 2.Uncoupling agents uncoupling protein (in brown adipose tissue), 2,4-dinitrophenol, Pentachlorophenol H + H + Ⅱ Ⅲ Intermenbran space Matrix uncoupling protein Q H + H + 2,4-dinitrophnol ADP+PiATP heat

Intermembrane space Matrix Oligomycin C ring 3.Oligomycin bonds at the connection of F 0 and F 1, inhibit the function of ATP synthase.

Oligomycin Ⅱ ⅠⅢ Ⅳ Succinate Ⅴ × × × ×× Uncoupling agent Retonone Amytal Antimycin A

ATP and other Energy-rich compounts ～ ～ ATP has two energy-rich phosphoric acid anhydride bonds, the hydrolysis of each bond release more energy than simple phosphate esters.

Some Energy-rich compounds ΔGº’ Structure Exemple phosphoenolpyruvate creatine phosphate acetyl phosphate Acetyl CoA

The hydrolysis of energy-rich bond: ΔG º’ = -5 ～ -15kcal/mol The compounds with energy-rich bond are high-energy compounds. The hydrolysis of low-energy bond: ΔG º’ = -1 ～ -3kcal/mol The compounds with low energy bond are low-energy compounds.

Transport of high-energy bond energies 1.Substrate level phosphorylation Glycerate 1,3-biphosphate + ADP Glycerate 3-phosphate +ATP ΔG º’ = -4.5kcal/mol Phosphoenolpyruvate +ADP Pyruvate + ATP ΔG º’ = -7.5kcal/mol

~P ~P ADP ATP Substrate level phosphorylation Oxidative Phosphorylation Energy utilization 2.ATP is the center of energy producing and utilizing.

3.Other nucleoside triphosphates are involved in energy transport. GTP: gluconeogenesis protein synthesis UTP: glycogen CTP: lipid synthesis

4.Transport of the terminal phosphate bond of ATP to the other nucleoside Function of nucleoside diphosphate kinase ATP + UDP ADP + UTP ATP + CDP ADP + CTP ATP + GDP ADP + GTP Function of adenylate kinase ADP + ADP ATP + AMP

7.3 Energy from cytosolic NADH 7.3 Energy from cytosolic NADH A mitochondrial NADH produce 2.5 ATP A cytosolic NADH must be transported into mitochondrial for oxidation by two methods. Glycerol phosphate shuttle 1.5 ATP Malate aspartate shuttle 2.5 ATP

Glycerol phosphate shuttle FADH 2 NAD + FAD Intermembran space Electron chain dihydroxyacetone phosphate Glycerol phosphate NADH+H + Glycerol phosphate dihydroxyacetone phosphate Inner menbran Glycerol phosphate dehydrogenase Glycerol phosphate dehydrogenase

NADH +H + NAD + NADH +H + NAD + Malate α-ketoglutarate carrier Glutamate-aspartate carrier oxaloacetate cytosol inner mitochondrial membran matrix Electron chain Aspartate Malate Aspartate Glutamate α-ketoglutarate oxaloacetate Malate aspartate shuttle

7.4 Other Biological Oxidations Monoxygenases dioxygenase --add 2 atoms of O 2 oxygenase to organic compounds. monoxygenase (mixed-function oxidase, hydroxylase) --adds 1 oxygen atom to organic compounds as a hydroxyl group. RH + NADPH + H + + O 2 ROH + NADP + + H 2 O

The chief compounds of monoxygenase: Cyt b 5, Cyt P 450, Cyt P 450 reductase(FAD,FMN)

Free Radical Scavenging Enzymes Free Radical: the groups with an unpaired electron. (such as O 2 ﹣ 、 H 2 O 2 、 OH) 1.Superoxide dismutases(SODs) 2O 2 ﹣ + 2H + H 2 O 2 + O 2 SOD H 2 O + O 2 peroxidase

2.Glutathione peroxidase Glutathion e peroxidase Glutathion e peroxidase H 2 O 2 (ROOH) H 2 O (ROH+H 2 O) 2G –SH G –S – S – G NADP + Glutathione reductase NADPH+H +

3.Catalase (in peroxisomes) 2H 2 O 2 2H 2 O + O 2 catalase

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