《生物化学—代谢篇》第二十四 章生物氧化

第24章生物氧化 电子传递与氧化礴化作用 eLectron transport and Oxidative phosphorylation

第24章 生物氧化 —电子传递与氧化磷酸化作用 Electron Transport and Oxidative Phosphorylation

Amino Fatty acids acid production Glycol Pyruvate pyruvate dehydrog co Acetyl-CoA Stage 2 分解代谢的三步曲 oxidation Citrate Oxaloacetate NADH FADH? (reduced stage 3 carnET Electron transfer and oxidative phosphorylation 2H+ Jo Respiratory (electron transfer) hain一 H. ADP+P ATP

一 分 解 代 谢 的 三 步 曲

●细胞内的线粒体 AT TP synthase Outer membrane F。F1) Freely permeable to Cristae 是生物氧化的主 small molecules and ions 要场所,主要功 Inner membrane 线 能是将代谢物脱 Impermeable to most mall molecules and ions 下的氢通过多种 酶及辅酶所组成 Respiratory electron carriers(Complexes I-IV) 的传递体系的传 ADP-ATP translocases . ATP synthase (FFu 递,最终与氧结 Other membrane 生成水。 Mat ●由供氢体、传递 Contains: Pyruvate 体、受氢体以及 dehydrogen 相应的酶催化系 Citric acid 组成 cycle enzymes 统组成的这种代 Fatty acid B-oxidation 谢途径一般称为 enzymes Amino acid oxidation 生物氧化还原链,m enzymes 当受氢体是氧时, Porin channels Many other enzymes 称为呼吸链。 ATP, ADP, P, Mg, Ca",K' Many intermediates

二线粒体呼吸链的组成  细胞内的线粒体 是生物氧化的主 要场所，主要功 能是将代谢物脱 下的氢通过多种 酶及辅酶所组成 的传递体系的传 递，最终与氧结 合生成水。  由供氢体、传递 体、受氢体以及 相应的酶催化系 统组成的这种代 谢途径一般称为 生物氧化还原链， 当受氢体是氧时， 称为呼吸链

NADH呼吸链 SH NADH CoQH2 Fe 脱氢酶 NADH脱氢酶 Cyt b K Cyt cI Cyt c yt a, NAD FMNH Fe 2H HO 2.FADH2呼吸链 MH FAD COQH F 黄素脱氢酶 Cyt bI Cyt CI Cyt c Cyt a, a M FADH2 Fe 2H. H,O

2. FADH2呼吸链 SH2 NADH NAD FMN FMNH2 CoQ CoQH2 Cyt b Cyt c1 Cyt c Cyt a, a3 O2 H2O Fe2+ Fe3+ NADH脱氢酶 Fe3+ Fe3+ Fe2+ Fe2+ Fe2+ Fe2+ 2H2+ 2 1 O - 脱氢酶 S CoQ CoQH2 Cyt b Cyt c1 Cyt c Cyt a, a3 O2 H2O Fe2+ Fe3+ Fe3+ Fe3+ Fe2+ Fe2+ Fe2+ Fe2+ 2H2+ 2 1 O - FAD FADH2 黄素脱氢酶 M MH2 1. NADH 呼吸链

、呼吸链各组份在线粒体內膜上的定位 现代生物化学认为,整个呼吸链由四个蛋白复合组分和两个游离的 成分组成: ★四个蛋白复合组分: Complexs I Complex、 Complex Ⅲ、 Complex Iv ★两个游离成分:辅醢Q和细胞色素C 67 72 Part Ill Bioenergetics and Catabolism intermembrane space(P side 4H' Cyt 吗Q lll 0+2H. NADH +H+ NAD+ Succinate Fumarate Matrix (N side)

三、呼吸链各组份在线粒体内膜上的定位 现代生物化学认为，整个呼吸链由四个蛋白复合组分和两个游离的 成分组成： ★ 四个蛋白复合组分：Complex Ⅰ、 Complex Ⅱ、 Complex Ⅲ 、Complex Ⅳ ★ 两个游离成分：辅酶Q和细胞色素C

四、呼吸链的电子传递在线粒体内膜两侧形成质 子梯度( A proton gradient across the inner membrane of mitochondria is generated using the electron motive force e An estimate of 10 protons are pumped for oxidizing one NADH and 6 for one FADh2 accompanying the electron flow through complexes I, III and iv e Conformational changes induced by electron transferring is believed to be coupled to proton pumping(however, the actual mechanisms is yet revealed e In actively respiring mitochondria, the measured Aph is about 0.75 and difference in electrical potential (Ap)is about 0.15-0.2V e The energy stored in such an H* gradient can be used to synthesize atP or to do other work

 An estimate of 10 protons are pumped for oxidizing one NADH and 6 for one FADH2 accompanying the electron flow through complexes I, III and IV.  Conformational changes induced by electron transferring is believed to be coupled to proton pumping (however, the actual mechanisms is yet revealed!).  In actively respiring mitochondria, the measured pH is about 0.75 and difference in electrical potential () is about 0.15-0.2 V.  The energy stored in such an H+ gradient can be used to synthesize ATP or to do other work. 四 、呼吸链的电子传递在线粒体内膜两侧形成质 子梯度（A proton gradient across the inner membrane of mitochondria is generated using the electron motive force ）

五、呼吸链顺序的实验证据( The order of the many electron carriers on the respiratory chain have been elucidated via various studies) o Measurement of the standard reduction potential(AE( Electrons tend to transfer from low E0 carriers to high E0 carriers (but may deviate from this in real cells) Oxidation kinetics studies: Full reduction followed by sudden O introduction, earlier oxidation, closer to the end of the respiratory chain; using rapid and sensitive spectrophotometric techniques to follow the oxidation of the cytochromes, which have different wavelength of maximal absorption Effects of various specific inhibitors: those before the blocked step should be reduced and those after be oxidized Isolation and characterization of each of the multiprotein complexes: specific electron donors and acceptors can be determined for portions of the chain

 Measurement of the standard reduction potential (E`0)): Electrons tend to transfer from low E`0 carriers to high E`0 carriers (but may deviate from this in real cells).  Oxidation kinetics studies: Full reduction followed by sudden O2 introduction; earlier oxidation, closer to the end of the respiratory chain; using rapid and sensitive spectrophotometric techniques to follow the oxidation of the cytochromes, which have different wavelength of maximal absorption.  Effects of various specific inhibitors: those before the blocked step should be reduced and those after be oxidized.  Isolation and characterization of each of the multiprotein complexes: specific electron donors and acceptors can be determined for portions of the chain. 五、呼吸链顺序的实验证据（The order of the many electron carriers on the respiratory chain have been elucidated via various studies）

六|。 atP would not be synthesized when 线 only adp and p; are added in isolated 与粒 mitochondria suspensions 体 eO consumption, an indication of 中 electron flow. was detected when a reductant(e.g, succinate) is added 呼 accompanied by an increase of ATP 成的偶联 吸| synthe esIs 链 e Both o consumption and atp it synthesis were suppressed when 子传递 inhibitors of respiratory chain(e.g cyanide, co, or antimycin A)was added

六 线 粒 体 中 呼 吸 链 电 子 传 递 与ATP 合 成 的 偶 联  ATP would not be synthesized when only ADP and Pi are added in isolated mitochondria suspensions.  O2 consumption, an indication of electron flow, was detected when a reductant (e.g., succinate) is added, accompanied by an increase of ATP synthesis.  Both O2 consumption and ATP synthesis were suppressed when inhibitors of respiratory chain (e.g., cyanide, CO, or antimycin A) was added

ATPsynthesis depends on the occurrence of electron flow in mitochondria O, consumption was neither observed ifADP was not added to the suspension, although a reductant is provided The o, consumption was also not observed in the presence of inhibitors of ATP synthase(e.g oligomycin or venturicidin) Electron flow also depends on ATP synthesis

 ATP synthesis depends on the occurrence of electron flow in mitochondria.  O2 consumption was neither observed if ADP was not added to the suspension, although a reductant is provided.  The O2 consumption was also not observed in the presence of inhibitors of ATP synthase (e.g., oligomycin or venturicidin).  Electron flow also depends on ATP synthesis!

七、电子传递与ATP合成的偶联机制( The chemiosmotic model was proposed to explain the coupling of electron flow and ATP synthesis) x First proposed in 1961 by Peter Michell(a British) x Energy released from electron transferring is hypothesized to be first used to pump protons from the mitochondrial matrix to the intermembrane space (or from stroma to thylakoid lumen in chloroplasts), thus generating a proton gradient across the inner membrane; such a proton-motive force then drives ATP synthesis by moving protons back into the matrix via the atP synthase-- The chemiosmotic model. x The model was initially opposed by virtually all researchers working in oxidative phosphorylation and photosynthesis

七、电子传递与ATP合成的偶联机制（The chemiosmotic model was proposed to explain the coupling of electron flow and ATP synthesis） •★First proposed in 1961 by Peter Michell (a British). •★ Energy released from electron transferring is hypothesized to be first used to pump protons from the mitochondrial matrix to the intermembrane space (or from stroma to thylakoid lumen in chloroplasts), thus generating a proton gradient across the inner membrane; such a proton-motive force then drives ATP synthesis by moving protons back into the matrix via the ATP synthase-- The chemiosmotic model. •★ The model was initially opposed by virtually all researchers working in oxidative phosphorylation and photosynthesis