Chapter 4. Biological Oxidation Intredction ATP oxidative phosphorylation Oxidation not producing ATP

biological oxidation introduction biological oxidation oxidation run in living body, detailedly, the process which nutrient substance, such as saccharides, lipids, and proteins are oxidized into water and carbon dioxide, and simultaneously　produce energy. Nutrient + O2  H2O + CO2 + energy ATP + heat

Characteristics of biological oxidation biological oxidation burning temperature 37℃ high condition neutral dry catalyst enzyme no Velocity of energy release slow fast Form of energy release ATP heat Way producing CO2and H2O organic acid decarboxylation produce CO2; wide addition of water and dehydrogenation , the hydrogen combine with oxygen at a indirect way. Oxygen directly combine with carbon(hydrogen), produce CO2(H2O).

Forms of oxidoreduction in biological oxidation oxidizing reaction loss of electrons dehydrogenation addation of oxygen reduction reaction Gain eletrons Addation of hydrogen Deoxygenation [O] [R] +e +H -H -e +O

General course of biological oxidation glucogen triglyceride protein glucose fatty acid + Glycerol amino acids AcetylCoA TAC CO2 ADP+Pi ATP 2H respiratory chain H2O

Energy of reaction G < 0 spontaneous G = 0 equilibrium G > 0　non-spontaneous G1 G2 G = G2-G1 G0’= - 2.303 R T log Keg = - n F E0’

Section 1. ATP 腺苷三磷酸 adenosine triphosphoric acid

activation effect

ATP is the main form of energy utilization and store in body and the center of energy conversion.

High-energy compound 分类及举例 释放能量（pH7.0,25℃) UTP、CTP、GTP 30.5 kJ/mol 1,3-二磷酸甘油酸、 磷酸烯醇式丙酮酸 61.9 kJ/mol 磷酸肌酸 43.9 kJ/mol 乙酰CoA、琥珀酰CoA、脂酰CoA 31.4 kJ/mol

nucleoside diphosphate kinase Transform　between high-energy compounds nucleoside diphosphate kinase GDP GTP ATP + UDP ADP + UTP CDP CTP adenylate kinase ADP + ADP  ATP + AMP

creatine phosphate– store form of ATP in brain and muscle.

Section 2. oxidative phosphorylation Ways producing ATP: substrate level phosphorylation oxidative phosphorylation substrate level phosphorylation --formation of ATP by the way of straight transfer high-energy substrate energy to ADP.

1,3-二磷酸甘油酸 + ADP 3-磷酸甘油酸 + ATP 琥珀酰CoA + H3PO4 + GDP 琥珀酸 + CoA-SH + GTP + ADP   ATP PK

I respiratory chain （呼吸链) a oxidoreduction system which consists of a series of enzyme, coenzyme aligning in mitochondrial inner membrane, function as linksystem transferer of hydrogen and electron.

Mitochondria Respiratory chain

Q electron transfer in respiratory chain e- e- e- e- e- H2O 内外膜间隙侧 基质侧 线粒体内膜 I Ⅱ Ⅲ Ⅳ Cytc e- e- e- e- e- H2O NADH+H+ 延胡素酸 1/2O2+2H+ NAD+ 琥珀酸

succinic acid oxidation respiratory chain NADH oxidation respiratory chain

Succinate-CoQ reductase Component of respiratory chain Complex name Number of peptide chain Prosthetic group ComplexⅠ NADH- CoQ reductase 42 FMN, Fe-S ComplexⅡ Succinate-CoQ reductase 4 FAD, Fe-S ComplexⅢ Q-Cyt C reductase 11 Fe-S, iron protoporphyrin, ComplexⅣ Cyt C oxidase 13 Cu, iron protoporphyrin,  Co-Q and Cyt C

(1) complexⅠ— NADH-Q reducase iron-sulphur protein, Flavoprotein with FMN, 42 peptide chains, 850 kD. Bind and oxidize NADH, transfer electrons to Q, release 4H+ to interspace of inner and outer membrane. NADH FMN，Fe-S CoQ

Structure of NAD+ and NADP+ R=H:NAD+; R=H2PO3:NADP+ NAD+:nicotinamide adenine dinucleotide,CoI NADP+:nicotinamide adenine dinucleotide phosphate,CoII

黄素单核甘酸

ironsulfur protein Fe-S Fe2+ Fe3+ + e Fe4S4 Fe2S2, Fe4S4

(Coenzyme Q,CoQ) 人体中： CoQ10 Ubiquinone,Q (Coenzyme Q,CoQ) 人体中： CoQ10 quinones contain a polyisoprene side chain. liposolubility，make it move in mitochondrial inner membrane easily. the only one electron carrier without protein in respiratory chain. 

2H+ FMN Fe-S N-2 Q QH2 复合体Ⅰ传递电子的过程 2H+ NAD+ NADH+H+ NADH+H+ FMN Fe2+ Q FMNH2 Fe3+ QH2 NAD+ Q NADH FMN Fe-S

Succinate-CoQ reductase (2) complexⅡ- Succinate-CoQ reductase i.e. succinate dehydrogenase, consists at list 4 peptides. Contain one FAD, two ironsulfur protein and one Cyt b560. Transfer electron from succinic acid to Q, do not release H+ to the interspace. succinic acid →FAD→Fe-S→Q。

cytochrome,Cyt 细胞色素 A、structure: colourant protein containing iron porphyrin. B、typing: Cyta: Cytaa3 Cytb: Cytb562 、Cytb566、 Cytb560 Cytc: Cytc 、 c1 C、difference: ① different side chain of iron porphyrin. Different linkage form of iron porphyrin with the protein. CytFe3+ + e  CytFe2+

甲酰基 多聚异戊二烯长链

Difference between Cyt a and Cyt b, Cyt c. prothetic group color αband wavelength Linkage with protein Cytb heme red 560nm Non-covalent bonding Cytc 550nm Bind with – SH of Cys Cyta heme A green 600nm Non-covalent bonding 　

(3) Complex Ⅲ Q-cytc reducase i.e. cyt c reducase, consists of 11 peptide chains different, existing as a dimer. every monomer contains two cyt b (b562, b566), one cyt c1 and a iron sulphur protein. Catalyze electron transfer from Q to cyt c. every two electrons’s transfering lead four proton pumped to the intermembrance space. QH2 Cytc b566b562Fe-Sc1

Cyt c Complex Ⅲ

Electron transfering process in complexⅢ first time oxidation of QH2 secondary time oxidation of QH2 Cytc Cytc Cytc1 2H+ Cytc1 2H+ Fe-S Fe-S e- e- bL bL e- QH2 Q e- QH2 Q bH bH Q  Q  Q QH2 2H+

(4) complex Ⅳ Cyt c oxidase Cyt c CuA a  a3  CuB O2 Dimer. Every monomer consists of 13 peptide chains different, as 3 subunits: I include 2 heme(a,a3),a cuproprotein (CuB);Ⅱinclude a dikaryon center formed by two copper ion(CuA);Ⅲ not clear Cu2+ + e  Cu+ Cyt c CuA a  a3  CuB O2

NADH氧化呼吸链 NADHFMN(Fe-S)Qbc1caa3O2 琥珀酸氧化呼吸链 succinic acidFAD(Fe-S)Qbc1caa3O2

overall reaction NADH + H+ + 1/2O2 NAD+ + H2O FADH2 + 1/2O2 FAD + H2O 呼吸链 or FADH2 + 1/2O2 呼吸链 FAD + H2O further 2H + 1/2O2 呼吸链 H2O In FADH2 or NADH

呼吸链中电子传递体的排列顺序的确定 利用脱氧胆酸处理线粒体内膜、分离出呼吸链的4种复合物，辅酶Q和细胞色素C及ATP合酶。 根据标准氧还电位E0’的高低排列 根据电子传递体氧化还原态时的吸收光谱变化进行检测 利用阻断剂研究分析 四种复合物的电子传递再造实验

II、oxidative phosphorylation oxidative phosphorylation refer to the ATP producing form which the reaction ADP change into ATP couple with respiratory chain oxidation. It is main form of ATP producing in body.

Why does the reaction: ADP + Pi  ATP + H2O request a couple? The product state(ATP+H2O) is higher energy level than reactant state(ADP + Pi). So, ADP change into ATP isn’t spontaneous process. Energy obtaining is requested for the process. ADP + Pi ATP+H2O 30.5kJ/mol energy energy level diagram

Which segment in respiratory chain can produce enugh energy for ADP phosphorylation？

What’s P/O ratio？What’s its meaning？ --- number of moles of ATP produced as consuming a mole of oxygen atom in a reaction, i.e. the number of moles of phosphor cosumed when consume a mole of oxygen atom in the reaction.

Chemiosmotic hypothesis What’s the way of ADP phosphorylation coupled with respiratory chain oxidation？ 氧化磷酸化偶联机制有：化学物质偶联学说、构象偶联学说及化学渗透学说。目前公认度较大的是化学渗透学说。 1961~1978 Chemiosmotic hypothesis the energy of respiratory chain oxidation change into proton gradient across the inner membrane. the proton gradient drive ATP-synase produce ATP.

In electron transfer process, respiratory chain put proton to intermembrance of inner and outer membrance, result in proton concentration different of the two side of the inner membrance.

conceptual diagram of Chemiosmotic hypothesis + + + + + + + + + + + + + + + + + + + - - - - - - - - - - - - - - - e- 延胡素酸 H2O NADH+H+ 琥珀酸 1/2O2+2H+ NAD+ ATP ADP+Pi

ATP synase Consists of hydrophobic F0(a1b2c912)and hydrophilic F1(33). When proton go straight through a,push c loop turning , and as a result, spur the F1 turning.

Position of ATP synase in mitochondria

work principle of ATP synase Three conformations of subunit:松L,紧T,放O Procedure of ATP production: 结好 （L） 脱水 （T） 松开 （O） H+ 

Ⅲ. Factors affecting oxidation phosphorylation Inhibitors Regulation by ADP Thyroid hormone Mitchondrial DNA mutation

(1)Inhibitors Inhibitors of respiratory chain block electron transfer of respiratory chain. 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 synase, like oligomycin

Blocking sites of inhibitor of respiratory chain CO、CN-、N3-及Ｈ２Ｓ 抗霉素Ａ 二巯基丙醇    异藤酮 粉蝶霉素Ａ 异戊巴比妥

Machanism of uncoupling Ｈ＋ 解偶联蛋白 热 Ｈ＋ Ｈ＋ ADP+Pi ATP+H2O

inhibitory action of oligomycin stop proton flow from F0 proton channel. 寡霉素

main regulation factor：ADP/ATP ratio (2) regulation by ADP main regulation factor：ADP/ATP ratio ADP + Pi  ATP + H2O Respiratory control ratio 离体线粒体实验,过量底物存在时,加入ADP后的耗氧速率与仅有底物时的耗氧速率之比

(3). Thyroid Hormone Thyroid Hormone 甲状腺激素 Na+-K+ ATP enzyme ATP degradation ADP/ATP Oxidative phosphorylation

(4) Mitchondrial DNA mutation function of mitochondrium Mitochondrium diseases all of the 13 peptides (7 peptides in NADH dehydrogenase, 1 in Cytc reducase, 3 in Cyt c oxydase,2 in ATP synase coded) by Mitochondrium join in oxidative phosphorylation. Mutation affect oxidative phosphorylation, ATP production decrease. Naked cyclic duble helix DNA, lack of defend system and restoration system. Symptoms are dependent on the degree of mutation and the different organs need for ATP. Maternally inherited diseases (heritage neurosis, heritage diabetes and deafness) Aging related

IV、 Mitochondria Entry and Exit of Molecules Mitochondrial porin, the major protein of the outer mitochondrial membrane, allows molecules less than 10 kD to pass Inner membrance were controled by differnet transporter.

Oxidation of NADH in cytosol α-glycerophosphate shuttle (α-磷酸甘油穿梭) malate-asparate shuttle (苹果酸-天冬氨酸穿梭)

Comparison of the two ways of NADH oxidation 氧化途径 主要存在的组织 主要承担酶 胞液中主要承担酶的辅基 线粒体内主要承担酶的辅基 被完全氧化时经过的呼吸链 完全氧化时产生的ATP量 -磷酸甘油穿梭 骨骼肌、神经细胞 α-磷酸甘油脱氢酶 NAD+ FAD 琥珀酸氧化呼吸链 2ATP 苹果酸穿梭 肝、心肌组织 苹果酸脱氢酶 NADH氧化呼吸链 3ATP

-glycerol phosphate shuttle 呼吸链 NADH+H+ FADH2 -磷酸甘油脱氢酶 -磷酸甘油脱氢酶 NAD+ FAD

malate-asparate shuttle 3ATP

Ⅰ. aerobic dehydrogenase and oxydase Section 3. other oxidation system Ⅰ. aerobic dehydrogenase and oxydase hydrogen acceptor prosthetic group product example 不需氧脱氢酶 辅酶 NADH脱氢酶 需氧脱氢酶 O2 FMN(FAD) H2O2 氨基酸氧化酶、 单胺氧化酶、黄嘌呤氧化酶 氧化酶 含Cu H2O 细胞色素c氧化酶、酚氧化酶 、抗坏血酸氧化酶

Ⅱ. Erzymes in peroxisome 过氧化酶体中的酶类 (1).catalase 过氧化氢酶 catalytic reaction: one molecule H2O2 offer electron;another molecule H2O2 accept electron. prosthetic group: 4 heme Function: wide distribution, wipe out toxical H2O2

(2). peroxidase （过氧化物酶） Catalytic reaction:catalyze H2O2 straight oxidize phenols and amines prosthetic group：1 heme Protect body. glutathione peroxidase Clinical diachorema occult blood test:

Ⅲ . superoxide dimutase, （ SOD,超氧化物歧化酶） O2-. H2O2 + .OH 损伤生物膜、生成脂褐素 SOD 2O2-. + 2H+ H2O2 + O2 H2O + O2 过氧化氢酶 SOD辅基含Cu、Zn(胞液) 或Mn(线粒体)。

(1). monooxygenase （加单氧酶） Ⅳ. Oxidases in microsome (1). monooxygenase （加单氧酶） Catalytic reaction: RH+NADPH+H++O2 ROH+NADP++H2O mixedfunction oxidase(混合功能氧化酶) or Hydroxylase(羟化酶). composition: NADPH-Cytc reducase, flavoprotein(FAD), ironsulfur protein(Fe2S2)、CytP450。 function：hydroxylation羟化。胆汁酸、胆固醇的生成；药物、毒物的转化；肾上腺皮质、类固醇激素的生物合成。

mechanism RH.P450.Fe3+ RH H2O ROH

(2). dioxygenases (双加氧酶) incorporate both oxygen atoms into the substrate. 色氨酸吡咯酶 O2

提要 ATP的生成主要通过氧化磷酸化。 呼吸链是线粒体内膜中的一系列递氢和递电子酶及其辅酶按照一定顺序排列成的连锁性氧化还原体系。主要有两条：NADHFMN(FeS)Qbc1c aa3  O2 琥珀酸FADH(FeS)Qbc1caa3O2 呼吸链的电子传递与氧化磷酸化有三处偶联：NADHQ；CtybCytc；Cytaa3O2 化学渗透假说 影响氧化磷酸化的因素 需氧脱氢酶、氧化酶、过氧化氢酶、过氧化物酶、SOD、羟化酶及加双氧酶的作用。

选择题练习 生物氧化

1. 呼吸链存在于（ ） A 细胞膜 B 线粒体外膜 C 线粒体内膜 D 微粒体 E 过氧化物酶体

2. 下列哪种物质不是NADH氧化呼吸链的组分？ A. FMN B. FAD C. 泛醌 D. 铁硫蛋白 E. 细胞色素c

3. ATP生成的主要方式是（ ） A 肌酸磷酸化 B 氧化磷酸化 C 糖的磷酸化 D 底物水平磷酸化 E 高能化合物之间的转化

4 由琥珀酸脱下的一对氢，经呼吸链氧化可产生（ ）分子ATP B 2 C 3 D 4 E 0

5 下例关于高能磷酸键的叙述，正确的是（ ） A 所有高能键都是磷酸键 B 高能磷酸键只存在于ATP C 高能磷酸键仅在呼吸链中偶联 D 有ATP参与的反应也可逆向进行 E 所有的生化转变都需要ATP参与

6. 下列哪种酶以氧为受氢体催化底物氧化生成水？ A 丙酮酸脱氢酶 B 琥珀酸脱氢酶 C 黄嘌呤氧化酶 D 细胞色素c氧化酶 E SOD

7. 关于线粒体内膜外H+浓度叙述正确的是( ) A 浓度高于线粒体内 B 浓度低于线粒体内 C 可自由进入线粒体 D 进入线粒体需主动转运 E 进入线粒体需载体转运

8. 参与呼吸链电子传递的金属离子是( ) A 铁离子 B 钴离子 C 镁离子 D 锌离子 E 以上都不是

9. 呼吸链中,不具有质子泵功能的是( ) A 复合体Ⅰ B 复合体Ⅱ C 复合体Ⅲ D 复合体Ⅳ E 以上都不是

10. 关于超氧化物歧化酶,哪项是不正确的( ) A 可催化产生超氧离子 B 可消除超氧离子 C 可催化产生过氧花氢 D 含金属离子辅基 E 存在于胞液和线粒体中

11. Except iron, Cyt aa3 contain ( ) ion. A Zn B Mg C Cu D Mn E K

12. Which one can be inhibited by CO in respiratory chain ? A FAD B FMN C Fe-S D Cyt aa3 E Cyt c

13. Which one is uncoupler? A CO B piericidin A C KCN D 2,4-dinitrophenol E H2S

14. The right electron tansferation sequence is ( ) A b→c→c1→aa3→O2 B c1→c→b→aa3→O2 C c→c1→b→aa3→O2 D c→b→c1→aa3→O2 E b→c1→c→aa3→O2

15. 关于ATP合成酶，叙述正确的是（ ） A 位于线粒体内膜，又称复合体Ⅴ B 由F1和F0两部分组成 C F0是质子通道 D 生成ATP的催化部位在F1的亚基上 E F1呈疏水性，嵌在线粒体内膜中

16. 关于辅酶Q, 哪些叙述是正确的? A 是一种水溶性化合物 B 其属醌类化合物 C 可在线粒体内膜中迅速扩散 D 不参与呼吸链复合体 E 是NADH呼吸链与琥珀酸呼吸链的交汇点

17. 关于细胞色素,叙述正确的是( ) A 均以铁卟啉为辅基 B 有色 C 均为电子传递体 D 均可被氰化物抑制 E 本质是蛋白质

18. 下列物质属于高能化合物的是（ ） A 乙酰辅酶A B GTP C 磷酸肌酸 D 磷酸二羟丙酮 E 磷酸烯醇式丙酮酸

19. Which make Fe-S as prosthetic group in the respiratory chain? A Complex Ⅰ B Complex Ⅱ C Complex Ⅲ D Complex Ⅳ E Cyt c

20. Where does the phosphorylation couple with the oxidation and can produce ATP? A NADH→CoQ B CoQ→Cyt b C CoQ→Cyt c D FADH2→CoQ E Cyt aa3→O2

论述题： 1 试述生物氧化与体外物质氧化的异同。 2 试述影响氧化磷酸化的因素及其作用机制。

名词解释： 呼吸链 氧化磷酸化。

谢谢！