生物化学和分子生物学：第8章 生物氧化.pptx

,目录,Biochemistry And Molecular Biology,Wei Yin PhD.,yinwei,Department of Biochemistry,Zhongshan Medical School,Sun Yat Sen University,2016-10-17,第,8,章,生 物 氧 化,Biological Oxidation,物质在生物体内进行氧化称,生物氧化,(biological oxidation),，主要指糖、脂肪、蛋白质等在体内分解时逐步释放能量，最终生成,CO,2,和,H,2,O,的过程。,糖,脂肪,蛋白质,CO,2,和,H,2,O,O,2,能量,ADP+Pi,ATP,热能,生物氧化的概念,生物氧化与体外氧化之相同点,生物氧化中物质的氧化方式有加氧、脱氢、失电子，遵循氧化还原反应的一般规律。,质在体内外氧化时所消耗的氧量、最终产物,(CO,2,，,H,2,O),和释放能量均相同。,反应环境温和，酶促反应逐步进行，能量逐步释放，能量容易捕获，,ATP,生成效率高。,通过加水脱氢反应使物质能间接获得氧，并增加脱氢的机会；脱下的氢与氧结合产生,H,2,O,，有机酸脱羧产生,CO,2,。,生物氧化与体外氧化之不同点,生物氧化,体外氧化,能量突然释放。,物质中的碳和氢直接氧结合生成,CO,2,和,H,2,O,。,糖原,三酯酰甘油,蛋白质,葡萄糖,脂酸,+,甘油,氨基酸,乙酰,CoA,TAC,2H,呼吸链,H,2,O,ADP+Pi,ATP,CO,2,生物氧化的一般过程,第一节 生成,ATP,的氧化磷酸化体系,The Oxidative Phosphorylation System with ATP Producing,指线粒体内膜中按一定顺序排列的一系列具有电子传递功能的酶复合体，可通过链锁的氧化还原将代谢物脱下的电子最终传递给氧生成水。这一系列酶和辅酶称为,呼吸链,(respiratory chain),又称,电子传递链,(electron transfer chain),。,一、呼吸链,定义,递氢体和电子传递体（,2H,2H,+,+2e,）,组成,酶复合体是线粒体内膜氧化呼吸链的天然存在形式，所含各组分具体完成电子传递过程。电子传递过程释放的能量驱动,H,+,移出线粒体内膜，转变为跨内膜,H,+,梯度的能量，再用于,ATP,的生物合成。,（一）氧化呼吸链由,4,种具有传递电子能力的复合体组成,呼吸链,各组分的分离,人线粒体呼吸链复合体,复合体,酶名称,质量,(kD),多肽链数,功能辅基,含结合位点,复合体,NADH-,泛醌还原酶,850,39,FMN,，,Fe-S,NADH,（基质侧）,CoQ,（脂质核心）,复合体,琥珀酸,-,泛醌还原酶,140,4,FAD,，,Fe-S,琥珀酸（基质侧）,CoQ,（脂质核心）,复合体,泛醌,-,细胞色素,C,还原酶,250,11,血红素,b,L,b,H,c,1,Fe-S,Cyt c,（膜间隙侧）,细胞色素,c,13,1,血红素,c,Cyt c,1,，,Cyt a,复合体,细胞色素,C,氧化酶,162,13,血红素,a,，,a,3,，,Cu,A,Cu,B,Cyt c,（膜间隙侧）,泛醌不包含在上述四种复合体中。,Overview of membrane-associated electron transport and ATP synthesis in mitochondria.,A proton concentration gradient is produced from reactions catalyzed by the electron transport chain.As electrons from reduced substrates flow through the complexes,protons are translocated across the inner mitochondrial membrane from the matrix to the intermembrane space.The free energy stored in the proton concentration gradient is utilized when protons flow back across the membrane via ATP synthase;their reentry is coupled to the conversion of ADP and Pi to ATP.,电子传递链及氧化磷酸化系统概貌：,1,、,复合体,作用是将,NADH+H,+,中的电子传递给泛醌,(ubiquinone),功能,:,将电子从,NADH,传递给泛醌,(ubiquinone,),NADH:,还原型烟酰胺腺嘌呤二核苷酸,(reduced nicotinamide adenine dinucleotide),组成：,黄素蛋白,(flavoprotein),含,FMN,铁硫蛋白,(iron-sulfur protein),含铁硫簇,FMN,：,黄素单核苷酸,(flavin mononucleotide),铁硫簇,(iro-sulofur cluster,Fe-S),复合体,将,NADH+H,+,中的两个电子传递给泛醌（,ubiquinone,）,复合体,NADH CoQ,FMN;Fe-S,N-1a,b,;,Fe-S,N-4,;,Fe-S,N-3,;Fe-S,N-2,NAD,+,和,NADP,+,的结构,R=H:NAD,+,;R=H,2,PO,3,:NADP,+,NAD,+,（,NADP,+,）,的加氢和脱氢反应,氧化还原反应时变化发生在,五价氮,和,三价氮,之间。,FMN,结构中含,核黄素,，发挥功能的部位是,异咯嗪环,，氧化还原反应时不稳定中间产物是,FMN,。在可逆的氧化还原反应中显示,3,种分子状态，属于,单、双电子传递体。,FMN,的加氢和脱氢反应,铁硫蛋白中辅基,铁硫中心,(Fe-S),含有等量铁原子和硫原子，其中一个铁原子可进行,Fe,2+,Fe,3+,+e,反应传递电子。,属于单电子传递体,。,表示无机硫,铁硫簇,(iron-sulfur cluster,),的结构,Fe,2,S,2,Fe,4,S,4,空间构象,Fe,铁硫簇,(iron-sulfur cluster,),的结构,泛醌（辅酶,Q,CoQ,Q,）由多个异戊二烯连接形成较长的疏水侧链（人,CoQ,10,），氧化还原反应时可生成中间产物,半醌型泛醌,。内膜中,可移动电子载体,，在各复合体间募集并穿梭传递还原当量和电子。在,电子传递和质子移动,的偶联中起着核心作用。,复合体,的功能,NADH+H,+,NAD,+,FMN,FMNH,2,还原型,Fe-S,氧化型,Fe-S,Q,QH,2,复合体,是三羧酸循环中的,琥珀酸脱氢酶,，又称,琥珀酸,-,泛醌还原酶,。,组成,:,黄素,蛋白（含,FAD,），,铁硫蛋白,细胞色素,(cytochrome,Cyt)b,560,电子传递,：,琥珀酸,FAD,几种,Fe-S CoQ,复合体,没有,H,+,泵的功能,。,2,、复合体,功能是将电子从琥珀酸传递到泛醌。,Structure of the,E.coli,succinate dehydrogenase complex,Complex II contains three identical multisubunit enzymes that associate to form a trimeric structure that is firmly embedded in the membrane.,A single copy of the enzyme showing the positions of FAD,the three FeS clusters,QH,2,and heme,b,.Complex II contains three copies of this multisubunit enzyme.,Electron transfer in Complex II,A pair of electrons is passed from succinate to FAD as part of citric acid cycle.Electrons are transferred one at a time from FADH,2,to three Fe-S clusters and then to Q.(Only one Fe-S cluster is shown in the figure.)Two protons are taken up from the interior to form QH,2,.Complex II does not directly contribute to the proton concentration gradient but serves as a tributary that supplies electrons(as)to the rest of the electron transport chain.,3,、复合体,功能是将电子从还原型泛醌传递给细胞色素,c,。,复合体,又叫,泛醌,-,细胞色素,C,还原酶,，细胞色素,b-c1,复合体，含有细胞色素,b(b562,b566),、细胞色素,c1,和一种可移动的铁硫蛋白,(Rieske protein),。,泛醌,从复合体,、,募集还原当量和电子并穿梭传递到复合体,。,电子传递过程：,CoQH2(Cyt bLCyt bH)Fe-S Cytc1Cytc,细胞色素,(cytochrome,Cyt),细胞色素是一类以,铁卟啉为辅基的催化电子传递的酶类,，根据它们吸收光谱不同而分类。,各细胞色素所含铁卟啉,(Cytb),(Cytc),(Cyta),Cytc,中铁卟啉,与蛋白质的连接,Complex III from cow(,Bos taurus,)mitochondria,Complex III contains two copies of the enzyme and is firmly anchored to the membrane by a large number of a-helices that span the lipid bilayer.The functional enzyme consists of three main subunits:cytochrome c,cytochrome,b,and the Rieske ironsulfur protein(ISP).,cytochrome,b,contains a heme group has a lower reduction potential(b,L,or,b,562,)and another heme has a higher reduction potential(b,H,or,b,566,),Complex III have two QH,2,binding site:Q,0,site(near the intermembrane space)and Q,i,site(near the matrix side),Electron transfer and proton flow in Complex III,Two pair of electrons are passed separately from two molecules of QH,2,to heme,b,L,(Q,0,site).Each pair of electrons is split so that individual electrons follow separate pathways.One electron is transferred to an Fe-S cluster,cytochrome,c,1,and finally to cytochrome,c,-the terminal electron acceptor.The other electron of each pair is transferred to heme,b,H,(Q,1,site)and then to Q.A total of four protons are translocated across the membrane:two from the inside compartment and two from QH,2.,Intermembrane,space,matrix,复合体,的电子传递通过,“,Q,循环”,实现。,复合体,每传递,2,个电子向内膜胞浆侧释放,4,个,H,+,，,复合体,也有质子泵作用,。,Cyt c,是,呼吸链唯一水溶性球状蛋白,，不包含在复合体中。将获得的电子传递到复合体,。,The Q cycle,During the oxidation of QH,2,to Q,one electron is donated tocytcvia a Rieske FeS and cytc1and the second to a Q to form the semiquinone via cytb,L,and cytb,H,with 2H,+,being released into the intermembrane space.A similar process then occurs with a second QH,2,but in this case the second electron is donated to the semiquinone,reducing it to QH,2,and 2H,+,are taken up from the matrix.(cyt,cytochrome;FeS,iron-sulfur protein;Q,coenzyme Q or ubiquinone.),The stoichiometryof the complete reaction,Four protons are produced during the oxidation of two molecules.These protons are released to the exterior of the membrane compartment and they contribute to the proton gradient that is formed during membrane associated electron transport.The protons originate in the interior compartment.They may have been taken up in the reactions catalyzed by complex I or complex II or they may be derived from protons taken up on the inside of the membrane during reduction of Q at the site in complex III.,Thus,for every pair of electrons that passes through complex III from QH,2,to cytochrome c,there are four protons translocated across the membrane.Two molecules of cytochrome c are reduced and these mobile carriers transport one electron each to complex IV.Note that there are actually two molecules of QH,2,oxidized(giving up four electrons)but two of these electrons are recycled to regenerate a molecule of QH,2,.,复合体,又称,细胞色素,C,氧化酶,(cytochrome c oxidase),。,电子传递：,Cyt cCuACyt aCyt a,3,CuBO,2,Cyt a,3,CuB,形成活性双核中心，将电子传递给,O,2,。每,2,个电子传递过程使,2,个,H,+,跨内膜向胞浆侧转移。,4,、复合体,将电子从细胞色素,C,传递给氧,Structure of cow(,Bos taurus,)complex IV from mitochondria,Subunit I is almost entirely embedded in the membrane.The bulk of this polypeptide consists of 12 transmembrane,-helices.There are three redox centers buried within subunit Itwo of them are,a,-type hemes(heme-,a,and a,3,),and the third is a copper atom Cu,B,.The copper atom is in close proximity to the iron atom of forming a binuclear center where the reduction of molecular oxygen takes place.,Subunit II has two transmembrane helices that anchor it to the membrane.Most of the polypeptide chain forms a,-barrel domain located on the exterior surface of the membrane.This domain contains a copper redox center(Cu,A,).composed of two copper atoms.These two copper atoms share electrons forming a mixed valence state.The external domain of subunit II is the site where cytochrome,c,binds to cytochrome,c,oxidase.,Complex IV is composed of two functional units of cytochrome,c,oxidase.Each unit is composed of 13 subunits with multiple membrane-spanning,-helices.In all species,each cytochrome,c,oxidase contains single copies of subunits I,II,and III.These polypeptides are encoded by mitochondrial genes in all eukaryotes.,复合体,的电子传递过程,细胞色素,c,氧化酶,CuB-Cyta3,中心使,O,2,还原成水的过程，有强氧化性中间物始终和双核中心紧密结合，不会引起细胞损伤。,The Protein Components of the,Mitochondrial Electron-Transfer Chain,and its Prosthetic Groups,The electron transport complexes can diffuse independently in the membrane but they tend to form larger structures through weak interactions with each other.The four enzyme complexes contain a wide variety of oxidationreduction centers.These may be cofactors such as FAD,FMN,or ubiquinone(Q).Other centers include FeS clusters,protein coenzymes such as the heme-containing cytochromes,and copper proteins.Electron flow occurs via the reduction and oxidation of these redox centers,with flow proceeding from a reducing agent to an oxidizing agent.,标准氧化还原电位,拆开和重组,特异抑制剂阻断,还原状态呼吸链缓慢给氧,（二）氧化呼吸链组分按氧化还原电位由低到高的顺序排列,由以下实验确定,:,The Sequence of Electron Carriers,Method 1:,Using standard reduction potentials of mitochondrial oxidationreduction components,The standard reduction potentials of the individual electron carriers have been determined experimentally.We would expect the carriers to function in order of increasing reduction potential,because electrons tend to flow spontaneously from carriers of lower,E,to carriers of higher,E,.The order of carriers deduced by this method is NADH Q cytochrome,b,cytochrome,c,1 cytochrome,c,cytochrome,a,cytochrome,a,3 O,2,.Note,however,that the order of standard reduction potentials is not necessarily the same as the order of,actual,reduction potentials under cellular conditions,which depend on the concentration of reduced and oxidized forms,.,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,E,0,(V),氧化还原对,E,0,(V),NAD,+,/NADN+H,+,0.32,Cyt c1 Fe,3+,/Fe,2+,0.22,FMN/FMNH,2,0.219,Cyt c Fe,3+,/Fe,2+,0.254,FAD/FADH,2,0.219,Cyt a Fe,3+,/Fe,2+,0.29,Cyt b,L,(b,H,)Fe,3+,/Fe,2+,0.05(0.10),Cyt a3 Fe,3+,/Fe,2+,0.35,Q,10,/Q,10,H,2,0.06,1/2O,2,/H,2,O,0.816,The Sequence of Electron Carriers,Method 2:,Observation of the state of oxidation or reduction of a given carrier within mitochondria,A second method for determining the sequence of electron carriers involves reducing the entire chain,of carriers experimentally by providing an electron source but no electron acceptor(no O,2,).When O,2,is,suddenly introduced into the system,the rate at which each electron carrier becomes oxidized(measured,spectroscopically)reveals the order in which the carriers function.The carrier nearest O,2,(at the end of the,chain)gives up its electrons first,the second carrier from the end is oxidized next,and so on.Such experiments,have confirmed the sequence deduced from standard reduction potentials.,Prosthetic groups of cytochromes,Each group consists of four five-membered,nitrogen-containing rings in a cyclic structure called a porphyrin.The four nitrogen atoms are coordinated with a central Fe ion,either Fe,2+,or Fe,3+,.Iron protoporphyrin IX is found in b-type cytochromes and in hemoglobin and myoglobin.Heme c is covalently bound to the protein of cytochrome c through thioether bonds to two Cys residues.Heme a,found in the a-type cytochromes,has a long isoprene tail attached to one of the five-membered rings.,The conjugated double-bond system(shaded pink)of the porphyrin ring accounts for the absorption of visible light by these hemes.,isoprene tail,Absorption spectra of cytochrome,c,(cyt,c,)in its oxidized(red)and reduced(blue)forms,Also labeled are the characteristic,and,bands of the reduced form.,The development a dual wavelength spectrophotometer permitted easy observation of the state of oxidation or reduction of a given carrier within mitochondria.This technique,together with the study of specific inhibitors,allowed some electron transport sequences to be assigned.,(a)Cytochrome,c,reduced spectrum;(b)cytochrome,c,oxidized spectrum;(c)the difference spectrum:(a)minus(b);(d)beef heart mitochondrial particles:room temperature difference(reduced minus oxidized)spectrum;(e)beef heart submitochondrial particles:same as(d)but at 77 K.,-and,-bands are labeled,and in(d)and(e)the bands for cytochromes,a,b,and,c,are indicated.,Typical visible absorption spectra of cytochromes,1,、,NADH,氧化呼吸链,NADH,复合体,Q,复合体,Cyt c,复合体,O,2,2,、琥珀酸氧化呼吸链,琥珀酸 复合体,Q,复合体,Cyt c,复合体,O,2,NADH,FMN,(Fe-S),琥珀酸,FAD,(Fe-S),CoQ,Cyt bCyt cCyt c,Cyt aa,3,O,2,NADH,氧化呼吸链,FADH,2,氧化呼吸链,第二节 氧化磷酸化,将氧化呼吸链释能与,ADP,磷酸化生成,ATP,偶联,氧化磷酸化,(oxidative phosphorylation),是指,在呼吸链电子传递过程中偶联,ADP,磷酸化，生成,ATP,，又称为,偶联磷酸化,。,底物水平磷酸化,(substrate level phosphorylation),与脱氢反应偶联，生成底物分子的高能键，使,ADP(GDP),磷酸化生成,ATP(GTP),的过程。不经电子传递。,ATP,生成方式,（一）氧化磷酸化偶联部位在复合体,、,、,内,根据,P/O,比值,自由能变化,:G,=-nFE,氧化磷酸化偶联部位：,复合体,、,、,线,粒,体,离,体,实,验,测,得,的,一,些,底,物,的,P/O,比,值,底,物,呼,吸,链,的,组,成,P/O,比,值,可,能,生,成,的,ATP,数,-,羟,丁,酸,NAD,+,复,合,体,CoQ,复,合,体,2.5,2.5,Cyt,c,复,合,体,O,2,琥,珀,酸,复,合,体,CoQ,复,合,体,1.5 1.5,Cyt,c,复,合,体,O,2,抗,坏,血,酸,Cyt,c,复,合,体,O,2,0.88,1,细,胞,色,素,c(Fe,2+,),复,合,体,O,2,0.61,-,0.68 1,1,、,P/O,比值,指氧化磷酸化过程中，每消耗,1/2,摩尔,O,2,所生成,ATP,的摩尔数（或一对电子通过氧化呼吸链传递给氧所生成,ATP,分子数）。,2,、自由能变化,根据热力学公式，,pH7.0,时标准自由能变化,(G0),与还原电位变化,(E0),之间有以下关系：,n,为传递电子数；,F,为法拉第常数,(96.5kJ/molV),G0=-nFE0,电子传递链自由能变化,ATP,ATP,ATP,氧化磷酸化偶联部位,NADH,FMN,(Fe-S),琥珀酸,FAD,(Fe-S),CoQ,Cyt bCyt cCyt c,Cyt aa,3,O,2,(,二,),氧化磷酸化偶联机制是产生跨线粒体内膜的质子梯度,1,、化学渗透假说,(chemiosmotic hypothesis),电子经呼吸链传递时，可将质子,(H,+,),从线粒体内膜的基质侧泵到内膜胞浆侧，产生膜内外质子电化学梯度储存能量。当质子顺浓度梯度回流时驱动,ADP,与,Pi,生成,ATP,。,氧化磷酸化依赖于完整封闭的线粒体内膜；,线粒体内膜对,H,+,、,OH,、,K,、,Cl,离子是不通透的；,电子传递链可驱动质子移出线粒体，形成可测定的跨内膜电化学梯度；,增加线粒体内膜外侧酸性可导致,ATP,合成，而线粒体内膜加入使质子通过物质可减少内膜质子梯度，结果电子虽可以传递，但,ATP,生成减少。,化学渗透假说已经得到广泛的实验支持。,线粒体基质,线粒体膜,+,-,H,+,O,2,H,2,O,H,+,e,-,ADP,+,Pi,ATP,化学渗透假说简单示意图,F,0,F,1,Cyt c,Q,NAD,H,+,H,+,NAD,+,延胡索酸,琥珀酸,H,+,1/2O,2,+,2H,+,H,2,O,ADP+Pi,ATP,4H,+,2H,+,4H,+,胞液侧,基质侧,+,-,电子传递过程,复合体,(4H,+,),、,(4 H,+,),和,(2H,+,),有质子泵功能,。,化学渗透示意图及各种抑制剂对电子传递链的影响,（三）质子顺梯度回流释放能量被,ATP,合酶利用催化,ATP,合成,F1,：亲水部分,（动物：,3,3,亚基复合体，,OSCP,、,IF1,亚基,），线粒体内膜的基质侧颗粒状突起，,催化,ATP,合成,。,F0,：疏水部分,（,ab,2,c,912,亚基，动物还有其他辅助亚基），镶嵌在线粒体内膜中，形成,跨内膜质子通道,。,ATP,合酶结构组成,Structure of,ATP Synthase,ATP,合酶组成可旋转的发动机样结构,F0,的,2,个,b,亚基的一端锚定,F1,的,亚基，另一端通过,和,33,稳固结合，使,a,、,b,2,和,3,3,、,亚基组成稳定的,定子部分,。,部分,和,亚基共同形成穿过,3,3,间中轴，,还与,1,个,亚基疏松结合作用，下端与嵌入内膜的,c,亚基环紧密结合。,c,亚基环、,和,亚基组成,转子部分,。,质子,顺梯度向基质,回流,时，转子部分相对定子部分旋转，使,ATP,合酶利用释放的能量,合成,ATP,。,当,H,+,顺浓度递度经,F,0,中,a,亚基和,c,亚基之间回流时，,亚基发生旋转,，,3,个,亚基的构象发生改变,。,ATP,合酶的工作机制,ATP,合成的结合变构机制,(binding change mechanism),F1 genetically engineered to contain a run of His residues adheres tightly to a microscope slide coated with a Ni complex;biotin is covalently attached to a c subunit of Fo.The protein avidin,which binds biotin very tightly,is covalently attached to long filaments of actin labeled with a fluorescent probe.Biotin-avidin binding now attaches the actin filaments to the c subunit.When ATP is provided as substrate for the ATPase activity of F,1,the labeled filament is seen to rotate continuously in one direction,proving that the Fo cylinder of c subunits rotates.,Rotation of F,o,and,experimentally demonstra