生化上课课件生物氧化

1、Biological Oxidation1Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10第六章生物氧化Chapter 6. Biological OxidationBiological Oxidation2Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Brief introduction

2、:5活细胞在执行细胞功能时，随时需要能量的补充，用于维持特定细胞结构、合成细胞成分、产生电流及其它过程等。5在绝大多数生物体内，能量的产生和储存的最主要途径是通过营养分子（糖、脂和蛋白质等）的彻底氧化（生成CO2和H2O）。在氧化的最终阶段，伴随 H 的氧化可将分解的大部分能量通过氧化磷酸化过程储存到 ATP 分子中。Biological Oxidation3Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Brief introduction

3、:5体内大多数氧化反应是以脱氢的形式发生的，在脱氢反应中，1 个或 2 个 H 原子(H+ + e)从底物脱下传递到氢接受体（主要是辅酶）。细胞内的氧化-还原反应涉及许多特异的电子传递体(electron carriers)。Biological Oxidation4Dr.Fucal Medicine School, SCU, 2006. 10Ø Catabolismof proteins,fats,andcarbohydrates in the three stagesof cellular respiration. Stage 1: oxidation of fatty acid

4、s, glucose, and some amino acids yields acetyl-CoA. Stage 2: oxidation of acetyl groups in thecitricacidcycleincludesfourstepsinwhichelectrons are abstracted. Stage 3: electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial (or, in bacteria, plasma membrane bound) electron car

5、riersthe respiratory chain ultimately reducing O2 to H2O. This electron flow drives the production of ATP.Biological Oxidation5Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Contents:6.1.16.1.26.1.36.1.4呼吸链氧化磷酸化氧化磷酸化的调节ATP 及其它“高能”化合物6.1.5 通过线粒体内膜

6、的物质转运6.1 生成 ATP 的氧化体系Oxidation System of ATP GenerationBiological Oxidation6Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Contents:1. 什么是呼吸链 ?2. 呼吸链的组成3. 呼吸链的排列6.1.1 呼吸链Respiratory ChainBiological Oxidation7Dr.Fu Qiang. Dept.of Biochemistry &

7、; Molecular Biology, Preclinical Medicine School, SCU, 2006. 101. 呼吸链的概念l 是存在于线粒内膜上的一系列蛋白、酶及辅助因子，它们组成相对独立的复合物并按一定顺序排列。营养物质氧化分解时产生的还原当量(-H 和电子) 经这些复合物一步步传递最终至 O2 生成 水，伴随 H 的氧化过程还可产生大量的 ATP。6.1.1 呼吸链Respiratory ChainBiological Oxidation8Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Precli

8、nical Medicine School, SCU, 2006. 102. 呼吸链的组成l 线粒体上的呼吸链由一系列顺序排列的电子传递体 (electron carriers) 所组成。这些电子传递体多数是线粒体内膜上的整合蛋白(integral proteins),其辅基可以传递 1 个或 2 个电子。l 呼吸链上的电子传递体通常以膜嵌合(membrane-embedded) 的方式形成几个相对独立的巨分子复合物 (supramolecular complexes) ，它们可以通过某些物理方法进行分离。Biological Oxidation9Dr.Fu Qiang. Dept.of Bi

9、ochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø 用去污剂(detergent) 温和地处理线粒体内膜后，可以得到 4 个单一的电子传递体复合物(electroncarrier complexes)，每个复合物分别可以在呼吸链中传递一部分电子。Biological Oxidation10Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 200

10、6. 10l 间于复合体间的两个游离成分: 泛醌和细胞色素cØ 复合体 I 和复合体 II 可分别将NADH 和琥珀酸(FADH2) 中的 H 和电子传递给泛醌(ubiquinone,或称辅酶Q, CoQ)。Ø 复合体III 将电子从还原型的泛醌(QH2) 传递给细 胞细胞 c (Cyt c)。Biological Oxidation11Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø Summary of t

11、he flow of electrons and protons through the four complexes of the respiratory chain. Electrons reach Q through Complexes Iand II. QH2 serves as a mobile carrier of electrons and protons. It passes electrons to Complex III, which passes them to another mobile connecting link, cytochrome c. Complex I

12、V then transfers electrons from reduced cytochrome c to O2. Electron flow through Complexes I, III, and IV is accompanied by proton flow from the matrix to the intermembrane space. Recall that electrons from oxidation of fatty acids can also enter the respiratory chain through Q.Biological Oxidation

13、12Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10(1). 复合体 I:NADH-泛醌还原酶(或 NADH 脱氢酶)l 复合体 I 是一个大分子酶复合物，至少含有 42个多肽链，这其中包括 1 个 含 FMN 的黄素蛋白和至少 6 个铁-硫中心(iron-sulfur centers), 或称铁-硫簇(iron-sulfur clusters)。l 在复合体 I 中进行的主要电子传递:NADH Æ FMN Æ Fe-S

14、Æ QBiological Oxidation13Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø Electrons and protons transferring in NADH: ubiquinone oxidoreductase (Complex I).Biological Oxidation14Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology,

15、 Preclinical Medicine School, SCU, 2006. 10Ø Oxidoreduction of NAD and NADP.Nicotinamideadenine dinucleotide, NAD,and its phosphorylated analog NADP undergo reduction to NADH and NADPH, accepting ahydride ion (two electrons and one proton) from an oxidizable substrate. The hydride ion is added

16、to either the front (the A side) or the back (the B side) of the planar nicotinamide ring.Biological Oxidation15Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø The transferring of protons and electrons via FMN.Flavoproteins containa very ti

17、ghtly, sometimes covalently, bound flavin nucleotide, either FMN or FAD. Theoxidized flavin nucleotide can accept either one electron (yielding the semiquinone form) or two (yielding FMNH2 or FADH2). Electron transfer occurs because the flavoprotein has a higher reduction potential than the compound

18、 oxidized.Biological Oxidation16Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø Iron-sulfur centers. TheFe-Scentersofiron-sulfur proteins may be as simple as (a), with a single Fe ion surrounded bythe S atoms of four Cys residues. Other cen

19、ters include both inorganic and Cys S atoms, as in (b) 2Fe-2S or (c) 4Fe-4S centers. (d) The ferredoxin of the cyanobacterium Anabaena 7120 has one 2Fe- 2S center (PDB ID 1FRD); Fe is red, inorganic S2 is yellow, and the S of Cys is orange. (Note that in these designations only the inorganic S atoms

20、 are counted. For example, in the 2Fe-2S center (b), each Fe ion is actually surrounded by four S atoms.) The exact standard reduction potential of the iron in these centers depends on the type of center and its interaction with the associated protein.Biological Oxidation17Dr.Fu Qiang. Dept.of Bioch

21、emistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10l Q 接受H 和电子后生醇(或称二氢泛醌, 即还原型的泛醌, QH2)，QH2 随即可在线粒体内膜上子传递给复合体 III，在这个的泵出。扩散并进一步将过程中会伴随Biological Oxidation18Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø Ubiquinone (

22、Q, or coenzyme Q). Completereduction of ubiquinone requires two electrons and two protons, and occurs in two steps through the semiquinone radical intermediate.Biological Oxidation19Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10l 复合体 I 催化两个偶联的反应

23、同时进行：c 转移NADH中的H: 和 1 个基质中的H+ 至泛醌，生成QH2（这是一个放能反应）：NADH + H+ +Q´ NAD+ + QH2l d 转移基质中的 4 个至线粒体内外膜间隙（吸能反应）：4HN´ 4HP+P for the positive side of the inner membrane (the intermembrane space), N for the negative side (the matrix).Biological Oxidation20Dr.Fu Qiang. Dept.of Biochemistry & Molec

24、ular Biology, Preclinical Medicine School, SCU, 2006. 10l 因此，复合体 I是一个质子泵(proton pump)，它利用电子传递反应释放的能量，定向地(vectorial)将质子从线粒体基质内泵出。NADH + 5HN+Q ´ NAD+QH2 + 4HP+P for the positive side of the inner membrane (theintermembrane spacN for the negative side (the matrix).Biological Oxidation21Dr.Fu Qiang

25、. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø Electrons and protons transferring in NADH:ubiquinone oxido- reductase (Complex I). Complex I catalyzes the transfer of a hydride ion fromNADH to FMN, from which two electrons pass through a series of Fe

26、-S centers to the ironsulfur protein N-2 in the matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses into the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons.Biological

27、Oxidation22Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Biological Oxidation23Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10(2). 复合体 II: 琥珀酸-泛醌还原酶(或 琥珀酸脱氢酶)l 复合体 II 含 4 个蛋白亚基及 5 个辅基(两类)。

28、16; 亚基A 和B 朝向基质侧，它们含有 3 个 2Fe-2S中心，结合 1 分子 FAD，以及 1 个底物(琥珀酸)结合位点。Ø 亚基 C 和 D 为膜整合蛋白，均含有 3 个跨膜螺旋，它们含有 1 个 血红素分子 (heme b) 以及 1个泛醌结合位点。Biological Oxidation24Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Structure of Complex II (succinate dehydr

29、ogenase) of E. coli. Theenzyme has two transmembrane subunits, C (green) and D (blue); the cytoplasmic extensions contain subunits B (orange) and A (purple). Just behind the FAD in subunit A (gold) is the binding site for succinate (occupied in this crystal structure by the inhibitor oxaloacetate, g

30、reen). Subunit B has three sets of Fe-S centers (yellow and red); ubiquinone (yellow) is bound to subunit C; and heme b (purple) is sandwiched between subunits C and D. A cardiolipin molecule is so tightly bound to subunit C that it shows up in the crystal structure (gray spacefilling).Electrons mov

31、e (blue arrows) from succinateto FAD, then through the three Fe-S centers to ubiquinone. The heme b is not on the main path of electron transfer but protects againstthe formation of reactive oxygen species (ROS) by electrons that go astray.¾Biological Oxidation25Dr.Fu Qiang. Dept.of Biochemistr

32、y & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10l 在复合体 II 上的电子传递途径是：首先从底物琥珀酸传递至FAD，生成FADH2，再经过Fe-S 中心传递给CoQ，生成还原型CoQ (QH2)。Ø 注: 血红素(heme b)在复合体II 上不参与电子传递。Biological Oxidation26Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006

33、. 10Biological Oxidation27Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10n 线粒体内的某些脱氢酶（如脂酰CoA 脱氢酶、3-磷酸甘油脱氢酶）催化的脱氢反应可以直接将底物的电子传递至CoQ（尽管其辅酶都是FAD）， 而不需要经过复合体 II。Biological Oxidation28Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclini

34、cal Medicine School, SCU, 2006. 10(3). 复合体 III: 泛醌-细胞色素c 还原酶(或 细胞色素bc1复合物)l 复合体 III 的作用是将电子从还原型泛醌(QH2)传递至细胞色素 c (Cyt c)。l 复合体 III 在传递电子的同时，会伴随将质子定向地从基质转移到线粒体外(内外膜间隙)。l 复合体 III 上的氧化还原的净反应为:QH2 + 2cyt c(Fe3+) + 2H´ Q + 2cyt c (Fe2+) + 4H+2NPBiological Oxidation29Dr.Fu Qiang. Dept.of Biochemistry

35、& Molecular Biology, Preclinical Medicine School, SCU, 2006. 10l 复合体 III 由两个相同单体组成二聚体(dimer)，每个单体由 11 个亚基构成。l 复合体 III 上的 3 个核心亚基：Ø 细胞色素b 及其两个血红素辅基(bH 和 bL);Ø Riske 铁-硫蛋其2Fe-2S中心；红素辅基Ø 细胞色素c1Biological Oxidation30Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinica

36、l Medicine School, SCU, 2006. 10Ø Structure of a Complex III monomer. The complex III is a dimer of identical monomers, each with 11 different subunits. The functional core is three subunits:cytochrome b (green) with its two hemes (bH and bL, light red); the Rieske iron-sulfur protein (purple)

37、with its 2Fe-2S centers (yellow); and cytochrome c1 (blue) with its heme (red).Biological Oxidation31Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10l 复合体 III 上的电子传递机制:Ø 泛醌循环(Q 循环) 提供了一个在双电子递体(CoQ)和单电子递体(Cyt b, c1, c)之间的一个转换(swith)。同时很好地解释了复合

38、体 III 也是一个 质子泵，每当一对电子经复合体 III 传递至Cyt c时，会伴随 4 个质子转出线粒体。Ø 尽管在复合体 III 上的电子传递机制很复杂，但净结果却很简单，即QH2 重新氧化成Q，2 分子Cyt c 被还原(Fe3+ ´ Fe2+)。Biological Oxidation32Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø The Q cycle. The path of electro

39、ns through Complex III is shown by blue arrows. On the P side of the membrane, two molecules of QH2 are oxidized to Q near the P side, releasing two protons per Q (four protons in all) into the intermembrane space. Each QH2 donates one electron (via the Rieske Fe-S center) to cytochrome c1, and one

40、electron (via cytochrome b) to a molecule of Q near the N side, reducing it in two steps to QH2. This reduction also uses two protons per Q, which are taken up from the matrix.Biological Oxidation33Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10B

41、iological Oxidation34Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10n 细胞色素 c (Cyt c) 是位于线粒体内外膜间隙的一个可溶性蛋白。当Cyt c 的血红素从复合体 III上获得 1 个电子后，它随即移向复合 IV，并向后者传递电子。Biological Oxidation35Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinica

42、l Medicine School, SCU, 2006. 10Ø Prosthetic groups of cytochromes. Each group consistsof 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 Fe2+ or Fe3+. Iron protoporphyrin IX is found in

43、 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 isoprenoid tail attached to one of the five-membered rings. The conjugated double- bond syst

44、em (shaded pink) of the porphyrin ring accounts for the absorption of visible light by these hemes.Biological Oxidation36Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10(4). 复合体 IV: 细胞色素c 氧化酶l 复体 IV 的作用是将还原型的Cyt c (Fe2+) 的电子传递给分子氧(O2)，并生成水。l 复合体 I

45、V 是一个位于线粒体内膜上的大分子酶复合物(含13 个亚基，Mr 204 kDa)。Ø 亚基 II 有一个双核中心 (CuA)，含 2 个铜离子，它们与两个Cys残基结合，这与铁硫蛋白的2Fe-2S 中心有些类似。Biological Oxidation37Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø 亚基 I2 个血红素个铜离子(C(分别命名为a 和 a3)，。血红素a3与CuB形成另以及另一个双核中心， 它们接受

46、血红素a 的电子并将其转移给与a3结合的O2。Ø 亚基 III 不参与电子传递，但其对复合体 IV 的功能完整性是必不可少的，其功能尚未完全阐明。Biological Oxidation38Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø Critical subunits of cytochrome oxidase (Complex IV). The bovine complex is shown here. (a)

47、The core of Complex IV, with three subunits. Subunit I (yellow) has two heme groups, a and a3 (red), and a copper ion, CuB (green sphere). Heme a3 and CuB form a binuclear Fe-Cu center. Subunit II (blue) contains two Cu ions (green spheres) complexed with the SH groups of two Cys residues in a binuc

48、lear center, CuA, that resembles the 2Fe-2S centers of iron-sulfur proteins. This binuclear center and the cytochrome cbinding site are located in a domain of subunit II that protrudes from the P side of the inner membrane (into the intermembrane space). Subunit III (green) seems to be essential for

49、 Complex IV function, but its role is not well understood. (b) The binuclear center of CuA. The Cu ions (green spheres) share electrons equally. When the center is reduced they have the formal charges Cu1+Cu1+; when oxidized, Cu1.5+Cu1.5+. Ligands around the Cu ions include two His (dark blue), two

50、Cys (yellow), an Asp (red), and Met (orange) residues.Biological Oxidation39Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10l 复合体 IV 上的电子传递方向是：Cyt c ´ CuAcenter ´ heme a ´ heme a3CuB center ´ O2.Biological Oxidation40Dr.Fu Qian

51、g. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10l 每当 4 个电子通过复合体 IV 传递时，该酶可消耗基质内(N侧)的 4 个H+ 用于生成水。同时，复合体IV 也可利用氧化还原的自由能将质子泵出至间隙(P侧)，每传递 1 个电子泵出 1 个质子。l 复合体 IV 上的总反应为：4Cyt c(Fe2+) + 8HN+ O2 ´ 4cyt c(Fe) + 4HP+ 2H2O+3+Biological Oxidation41Dr.Fu Qiang. De

52、pt.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø PathofelectronsthroughComplex IV. Thethreeproteinscritical to electron flow are subunits I, II,andIII.Thelargergreenstructureincludes the other ten proteins in thecomplex.ElectrontransferthroughComplex IV be

53、gins when two molecules of reduced cytochrome c (top) each donate an electron to the binuclear center CuA. From here electrons pass throughheme a to the Fe-Cu center (cytochromea3 and CuB). Oxygen now binds to hemea3 and is reduced to its peroxy derivative(O22) by two electrons from the Fe-Cu2center

54、. Delivery of two more electronsfromcytochromec(makingfourelectrons in all) converts the O22 to two2molecules of water, with consumption of four “substrate” protons from the matrix. At the same time, four more protons are pumped from the matrix by an as yet unknown mechanism.Biological Oxidation42Dr

55、.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Biological Oxidation43Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 103. 呼吸链组分的排列顺序l 在呼吸链催化的整个反应中，电子从 NADH，琥珀酸或其它底物经黄素蛋白、泛醌、铁-硫蛋白、细胞色素c，最后传至O2。l 呼吸链

56、各组分排列顺序的确定使用了多种方法:Ø 标准还原电位(E°) ：先对各电子传递体的标准还原电位进行测定，再按由低到高的顺序排列(因为电子总是倾向自发地从低电位向高电位移动)。Biological Oxidation44Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Ø 按氧化还原电递链排列应为:低到高的顺序推导的电子传Cyt b Cyt c1 Cyt c Cyt a NADH QCyt a3 O2Biological Oxidation45Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Preclinical Medicine School, SCU, 2006. 10Biological Oxidation46Dr.Fu Qiang. Dept.of Biochemistry & Molecular Biology, Pr