Chapter 7 Mitochondria.2017sF ppt(1).pptx

CHAPTER 7,Mitochondria and Peroxisome线粒体和过氧化物酶体线粒体和过氧化物酶体,The research of mitochondrial functionEarly in 1850,Klliker:osmotic phenomenon.In 1900，Michaelis,Janus green B(詹纳斯绿),liver cells:oxygen:Indicator blueIn 1913，Warburg separated mitochondria from cell homogenate(细胞匀浆)and discovered that it could consume o2.,Early in 1940s，Claude invented the isolation technique,obtained mitochondria using salt extraction techniques.In 1948,George Hogeboom,Walter Schneider and George Palade obtained activated mitochondria using sugar extraction techniques.Kenedy and Lehninger:tricarboxylic acid cycle（三羧酸循环），electron-transport，oxidative phosphorylation（氧化磷酸化），fatty acid oxidation(脂肪酸氧化).in 1950s，G.Palade,K.Porter and F.Sjostrand等-electron microscope:ultrastructure(超微结构),OUTLINE,Mitochondria structure Mitochondria protein localization Mitochondria function-oxidative phosphorylation Mitochondria proliferation Peroxisomes,1.Structure of Mitochondria(1)Shape:Granular(粒状),bacilliform(杆状)or elongated cylinders with a diameter of 0.51.0m.,(2)The number and distributionNumber:obvious difference in different types of cells:1000-2000 Mi in liver cells and no Mi in mammal mature red blood cells.Distribution:the regions requiring energy:fat droplet and sperm tail.,鞭毛轴丝,肌原纤维,(3)Ultrastructure of Mitochondria,（外室）,（内室）,Outer membrane Lipid/protein(6 nm thick):1:1-rich porins（孔蛋白）forming large water-filled channels:like a sieve permeable to all hydrophilic molecules of less than 5 k Da.,TOM complexes（translocator complexes of outer membrane）and some special enzymes.Marker enzyme-Monoamine oxidase(单胺氧化酶):catalyzing oxidative deaminization(氧化脱氨)of monoamine(单胺).,Inner membrane（6-8 nm thick）Lipid/protein:-high proportion of cardiolipin(心磷脂):the membrane impermeable to ions.usually highly convoluted-forming a series of infoldings(cristae-嵴)projecting into the matrix and covered with many granum(基粒：基本颗粒,elementary particle).,双磷脂酰甘油,many types of proteins for energy conversion:electron-transport chain,ATP synthase.Marker enzyme-cytochrome oxidase(细胞色素氧化酶),Many types of transport proteins:metabolites into and out of the matrix，TIM complexes（translocator complexes of inner membrane-内膜转运蛋白复合体）,Intermenbrane space（6-8 nm wide）many soluble enzymes,substrates and cofactors-set up electrochemical gradient.Marker enzyme-adenylatekinase(腺苷酸激酶):Formation of ADP AMP+ATP2ADP,Matrix Highly concentrated mixture of hundreds of enzymes-the oxidation of pyruvate,fatty acids and amino acids.Marker enzyme-malate dehydrogenase(苹果酸脱氢酶):苹果酸 草酰乙酸.,mitochondrial genome,ribosomes,tRNAs and various enzymes for gene expression.dense granular deposits of calcium phosphate(磷酸钙)-Ca2+storage,线粒体组分的分离,毛地黄苷,线粒体质体,(芦布若尔：膜蛋白的增溶剂),(4)Chemical component of Mi,proteins(65%-70%)and Lipids(25%-30%-Phospholipids(Principal ingredient).-Inner membrane:P/L(80/20),Inner membrane proteins(21%Mi proteins)-Outer membrane:P/L(50/50),outer membrane proteins(6%Mi proteins)-Intermenbrane space:6%Mi proteins-Matrix:67%Mi proteins(enzymes,Coenzyme,vitamins,and metal ions，DNA，RNA and ribosomes）,2.Mitochondria protein targeting,Cell address target(地址签),1)Translocation Mi protein target,Leading peptide（导肽或引导肽）:位于新生肽链N-末端的一段氨基酸序列，能引导肽链进入线粒体。(1)Characteristics(特征)Sequence characteristics-at the N-terminus-about 20-80 amino acid sequence in length-rich alkaline amino acids(Arginine and lysine)-few acidic amino acids-positively charged amino acids alternate with hydrophobic ones in signal sequences,an amphiphilic a helix(hydrophobic and hydrophilic):specific receptor recognizes this configuration rather than the precise amino acid sequence.,subunit of cytochrome oxidase,In the leading peptide,different sequences contain different information.,双导向序列,The leading peptide has no specific claim on the guided proteins.,实验设计分离线粒体与具有线粒体基质定位信号的前体蛋白温育胰蛋白酶处理,导肽如何引导蛋白质进入线粒体?,Precursor proteins unfolded Many types of translocators involved in the process:,(2)Translocation of mitochondrial proteins,Translocase of the outer and inner membranes,The matrix protein translocation,The inner membrane protein translocation,OXA负责将基质中线粒体DNA编码的内膜蛋白和被运进基质的内膜蛋白插到内膜上,two Routes,A family of metabolite-specific transporters for ATP,ADP,and phosphate,which are multipass transmembrane proteins.The TIM22 complex mediates the insertion of these types of proteins.,.,The intermembrane space protein translocation,(a)conservative sorting,transiently bind specialized chaperone proteins,both inserts proteins into the outer membrane and helps them fold properly.,First into the intermembrane space across the TOM complex,The outer membrane protein translocation,The N-terminal amino acids of the proteins in the outer membrane can recognize receptor in the outer membrane.,(3)The requisite factor for translocation of Mi proteins,leading peptide（导肽）chaperone proteins(分子伴侣)receptor（受体）the contact point（接触点）between the outer membrane and inner membrane energy(ATP and Membrane Potential)transit peptidase(转运肽酶)translocators（膜转运复合体）,3.Mi Function,Oxidative phosphorylation(氧化磷酸化)产生ATP Regulation of cytosolic Ca2+concentration Regulation of the cell apoptosis,Aerobic oxidation of glucose:Glycolysis(糖酵解:细胞质)pyruvate(丙酮酸)acetyl CoA(pyruvate dehydrogenase):mitochondrial matrix)Tricarboxylic acid cycle(三羧酸循环-TCA cycle).,乳酸,（1)oxidation（生物氧化）：细胞质中的多糖、脂肪和蛋白质分解成简单的可溶性产物）-细胞氧化,Oxidative phosphorylation,视频,Krebs:英国生化学家:32岁-鸟氨酸循环;37岁 TCA:三种主要有机物代谢的联系枢纽,1953获诺贝尔生理医学奖.,TCA cycle:tricarboxylic acid cycle(三羧酸循环),Krebs cycle,柠檬酸循环,柠檬酸有3个羧基,Oxidation of reductive coenzyme-oxidation of glucose produced NADH and FADH2.,黄素腺嘌呤二核苷酸,烟酰胺腺嘌呤二核苷酸,NAD+NADH FAD+FADH2,NADH+1/2 O2 NAD+能量 FADH2+1/2 O2 FAD+能量,NADH和FADH2必须被氧化才能release energy,NADH和FADH2如何被氧化及被氧化时释放的H+、电子和能量如何安置?,Each complex has an absorption spectrum，which can absorb visible light and change color when they are oxidized or reduced.Many complexes were isolated and identified from inner membrane.Each of these purified complexes can be inserted into lipid bilayer vesicles.,离子交换色谱法,Function:H+and electron transfer,many complexes,Three large enzyme complexes embedded in the inner membrane and one small complex.The complexes are asymmetrically oriented in the inner membrane.,复合体 的晶体结构被中国的饶子和院士团队,和 分别为美国和日本解析，复合体 I还不清楚,Electron-transport(电子传递）,Electron-transport chain(电子传递链)-Respiratory chain(呼吸链)：A series of protein complexes arranged in order(按顺序排列)in the Mi inner membrane,can reversibly accept or release H+and electrons.,功能是参与对还原型辅酶的氧化,(A)NADH dehydrogenase complex(NADH脱氢酶复合体):the largest complex(more than 40 polypeptide chains),a flavin prosthetic group(辅基),at least seven iron-sulfur centers,Complex I,Complex I catalyzes NADH oxidation by accepting two high-energy electrons from NADH and passing them to ubiquinone,transferring H+from the matrix to the intermembrane space.,NADH-coenzyme Q reductase,Complex I,黄素单核苷酸,(B)Succinate dehydrogenase(琥珀酸脱氢酶):a FAD prosthetic group,two iron-sulfur proteins.,Complex,Complex transfers two low-energy electrons to CoQ from succinate(琥珀酸+Q延胡索酸+QH2).,Succinate-coenzyme Q reductase(琥珀酸-coenzyme Q 还原酶),黄素腺嘌呤二核苷酸,(C)Cytochrome b-c1 complex(细胞色素b-c1复合体):at least 11 different polypeptide chains，a dimer.Each monomer：one cytochrome b(two hemes),one cytochrome c1(one heme),an iron-sulfur protein.,Complex,Complex accepts electrons from ubiquinone and passes them on to cytochrome c，and pumps proton from the matrix to the intermembrane space.,CoQ-cytochrome c reductase,（D）cytochrome c oxidase complex（细胞色素c氧化酶复合体):a dimer,each monomer:l3 different polypeptide chains,two cytochromes（cyta and cyta3）and two copper(Cu)atoms.,Complex,It accepts one electron at a time from cytochrome c and passes four electrons at a time to oxygen,It is here that nearly all of the oxygen(90%)we breathe is used.,4cyt(还原型)+8HN+O2 4cyt(氧化型)+4Hp+2H2O,Order of Respiratory Chain Complexes,线粒体内膜主次呼吸链,NADH respiratory chain,Succinate respiratory chain,NADH respiratory chain-主呼吸链 Succinate(琥珀酸)respiratory chain-次呼吸链 不经,Electron carriers（递电子体）,Four types of electron carriers：flavinprotein(黄素蛋白)cytochromes(细胞色素)iron-sulfur proteins(Fe-S蛋白)ubiquinone(泛醌)or coenzyme Q(辅酶Q).-The flavinproteins and coenzyme Q also transfer H+,Flavinprotein：NADH-coenzyme Q reductase with a prosthetic groups of FMN;succinate-coenzyme Q reductase with a prosthetic groups of FAD Accepting two electrons and two protons.,Cytochromes(细胞色素),Each cytochrome molecule consists of a chromoprotein(色素蛋白)and a prosthetic groups of ferriporphyrin(铁卟啉)that is attached covalently to the protein.there are five types of cytochromes of a,a3(a,a3除含铁外，还含铜),b,c,c1，which transfers electrons by the conversion between Fe3+and Fe2+by passing one electron at a time.,血红素铁,Cytochrome c is a small,water-soluble protein consisting of a 104-AA polypeptied chain with a single heme group.,The molecule contains sulfurs and prosthetic groups of non-heme irons(非血红素铁)linked with cysteine side chains,.forming an iron-sulfur center within the protein.Like the cytochromes,these centers carry one electron at a time by the conversion between Fe3+and Fe2+.,Fe3+e-Fe2+,iron-sulfur proteins(Fe-S):,Two common forms:Fe2S2 and Fe4S4,Coenzyme QUbiquinone(泛醌)：A quinone(Q)is a small hydrophobic(脂溶性)molecule that can pick up or donate either one or two electrons.,ATP synthesis is coupled to electron transport,(2)ATP synthesis,线粒体内膜重建,超声波破碎,亚线粒体颗粒（sub-mitochondrial particle）,the site of ATP synthesis.,the site of electron transport,Proton gradient（pH）Potential difference(内膜两侧电位差-膜电位),Institution of transmembrane electrochemical gradient(质子动力),主要作用,Transmembrane electrochemical gradient drives ATP synthesis in the presence of ADP,Pi and F0-F1-ATPase(F0-F1 coupling factorF0-F1偶联因子).,ATP synthase,ATP synthase or F0-F1-ATPase(H+-ATPase):widely distributed in the membrane of mitochondria,chloroplast,heterotrophic bacterium(异养菌)and photosynthetic bacterium,involved in ATP synthesis.,mushroom-like,ATP synthase embedded in cristae(嵴)consist of a head portion(F1)and a transmembrane H+carrier(F0)F1 catalytic subunit:five kinds of polypeptides 33.Each subunit has a catalytic site of ATP synthesis.F0 complex:three types of subunits(1a,2b,10-14c),mediates proton transport.A rotating stalk(and)is fixed to a rotor.F1 and F0 are linked by the rotor and the stator,rotor of c ring,stator of,a,b,Structure of ATP synthase,寡霉素（oligomycin）可与F0结合，阻塞质子通道，使ATP synthase 失去活性。,1960s，美国学者Paul Boyer:binding-change model（结合变构模型：ATP合成酶合成ATP的机制）,1、F1上有3个活性部位轮替催化合成ATP，亚基开始处于-ADP构象，可与介质中的ADP和磷酸结合2、亚基转变为-ATP构象，与ATP紧密结合。3、亚基最后转变为-排空构象。ATP离开酶表面。4、亚基中心轴每旋转120度，即与不同的亚基接触，迫使其变为-排空构象。,Proton flow C unit rotates rotates conformation change ATP synthesized,In 1994,Walker JE published the crystal structure of bovine(牛心)mitochondrial F1-ATPase and obtained Nobel Prizes in Chemistry with Boyer in 1997.,Function of F0F1particles F0F1particle is a kind of reversible multiplex enzyme ATPase:ATP hydrolysis ATP synthase(ATP合酶):ATP synthesis,-Chemiosmotic coupling hypothesis（化学渗透学说，1961,Peter Mitchell,a britain biochemist):electron transport-pump protons across the membrane-electrochemical proton gradient-protons flows back through F0F1 particles-ATP synthesis.,ATP synthesis,The hypothesis may explain the relationship between electron transport,generation of proton electrochemical gradient and ADP phosphorlation.Therefore,he obtained Nobel Prizes in Chemistry in 1978.Oxidative phosphorylation(氧化磷酸化):A process of the synthesis of ATP coupled with respiratory chain oxidation in Mi.底物水平的磷酸化,(A)Inhibiter of electron transport(B)Inhibiter of proton transport(C)Uncoupling agent(解偶联剂):a compound that disrupts the usual tight coupling between electron transport and ADP phosphorylation.Uncouplers block oxidative phosphorylation by dissipating the H+electrochemical gradient,