生化课件Oxidative Phosporylation

1、Section L Respiration and energy,Electron transferring via a chain of membrane bound carriers, across membrane proton gradient, ATP synthesis (with O2 consumed)- Oxidative phosphorylation,Cells need energy to do all their work 2 ATP is the universal currency for biological energy,This was first perc

2、eived by Fritz Lipmann and Herman Kalckar in 1941 when studying glycolysis. Hydrolysis of the two phosphoanhydride (磷酸酐键) bonds in ATP generate more stable products releasing large amount of free energy (Go is -30.5 kJ/mol; G in cells is -50 to -65 kJ/mol).,The ATP molecule is kinetically stable at

3、pH 7 (i.e., it has a high activation energy, G for hydrolysis) and enzyme catalysis is needed for its hydrolysis. ATP is not a long-term storage form of free energy in living cells, being consumed within a minute following its formation (phosphocreatine, 磷酸肌酸, act as a energy storage form for longer

4、 term). A resting human consumes about 40 kg of ATP in 24 hours!,ATP has an intermediate phosphoryl group transfer potential.,3 Electron transfer via redox reactions generates biological energy,When electrons flow from a low affinity carrier (reductant) to a high affinity carrier (oxidant), either i

5、n an electric battery or in a living cell, energy is released and work can be done. Oxidation of energy-rich biological fuels often means dehydrogenation (catalyzed by dehydrogenases, 脱氢酶) from carbons having various oxidation states. In the living cells, electrons are transferred directly as electr

6、ons (between metal ions), as hydrogen atoms (H+e-), or as a hydride ion (:H- or H+2e-).,Energy is “generated” via electron flow both in a battery and in a cell!,4 Oxidative phosphorylation,Oxidative phosphorylation is the culmination of energy-yielding metabolism in aerobic organisms. All oxidative

7、steps in the degradation of carbohydrates, fats, and amino acids converge at this final stage of cellular respiration, in which the energy of oxidation drives the synthesis of ATP.,In eukaryotes, oxidative phosphorylation occurs in mitochondria. Oxidative phosphorylation involves the reduction of O2

8、 to H2O with electrons donated by NADH and FADH2; it occurs equally well in light or darkness.,5 The electron transport chain involves several different molecular species, Various prosthetic groups act as intermediate electron carriers in the respiratory chain:,(a) Flavoproteins, which contain tight

9、ly bound FMN or FAD as prosthetic groups, and which may participate in one- or two-electron transfer events. (b) Coenzyme Q, also called ubiquinone (and abbreviated CoQ or UQ), which can function in either one- or two-electron transfer reactions.,(c) Several cytochromes (proteins containing heme pro

10、sthetic groups, which function by carrying or transferring electrons), including cytochromes b, c, c1, a, and a3. Cytochromes are one-electron transfer agents, in which the heme iron is converted from Fe2+ (ferrous)to Fe3+ (ferric) and back. (d) A number of ironsulfur proteins, which participate in

11、one-electron transfers involving the Fe2+ and Fe3+ states. (e) Protein-bound copper, a one-electron transfer site, which converts between Cu+ (cuprous) and Cu2+(cupric ).,Nicotinamide is derived From niacin(B5),FMN (Flavin MonoNucleotide) FAD (Flavin Adenine Dinucleotide),NAD+, NADP+, FAD are the fe

12、w commonly used such reversible electron carriers. NAD and NADP are dinucleotides able to accept/donate a hydride ion (H- , thus with 2e-) for each round of reduction/oxidation. Reduction of NAD+ and NADP+ can be easily followed by spectroscopy (at 340 nm).,FAD is able to accept/donate one or two el

13、ectrons (as hydrogen atom), with absorption maximum shifts from 570 nm to 450nm. NAD and NADP can easily diffuse out of the enzymes, but FMN and FAD are tightly bound to the enzymes. NADH and FADH2 will be further oxidized via the respiratory chain on the inner membrane of mitochondria or plasma mem

14、brane of bacteria for energy generation (transduction).,Q is a mobile one/two e-/H+ carrier,The only e- carrier not bound to a protein,Q/QH2 diffuses freely in the lipid bilayer,Three types of heme groups are found in cytochromes.,At least eight different types of iron-sulfur centers act in the resp

15、iratory chain: iron atoms cycle between Fe2+ (reduced) and Fe3+ (oxidized).,Iron-sulfur centers: The Fe-S centers of iron-sulfur proteins may be as simple as (a), with a single Fe ion surrounded by the S atoms of four Cys residues. Other centers include both inorganic and Cys S atoms, as in (b) 2Fe-

16、2S or (c) 4Fe-4S centers.,6 Electrons of NADH and FADH2 are transferred to O2 via many intermediate electron carriers on the way.,The respiratory chain be considered to be composed of four parts:,(I) NADHcoenzyme Q reductase (II) succinatecoenzyme Q reductase (III) coenzyme Qcytochrome c reductase (

17、IV) cytochrome c oxidase, 10 protons are pumped per NADH, and 6 per FADH2 oxidized via the respiratory chain.,7 The order of the many electron carriers on the respiratory chain have been elucidated via various studies,First, the standard reduction potentials of the individual electron carriers have

18、been determined experimentally. Because electrons tend to flow spontaneously from carriers of lower E0 to carriers of higher E0.,Electron carriers may have an order of increasing E0,A second method for determining the sequence of electron carriers involves reducing the entire chain of carriers exper

19、imentally by providing an electron source but no electron acceptor (no O2). When O2 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 O2 (at the end of th

20、e chain) gives up its electrons first, the second carrier from the end is oxidized next, and so on.,In a final confirmation, agents that inhibit the flow of electrons through the chain have been used in combination with measurements of the degree of oxidation of each carrier. In the presence of O2 a

21、nd an electron donor, carriers that function before the inhibited step become fully reduced, and those that function after this step are completely oxidized.,Studies with specific inhibitors help to reveal the orders of the e- carriers,Reduced,Oxidized,Reduced,Oxidized,Reduced,8 Electron transfer to

22、 O2 was found to be obligatorily coupled to ATP synthesis from ADP + Pi in isolated mitochondria,How is oxidation of NADH/FADH2 coupled to the phosphorylation of ADP?,The P/O ratio,The number of molecules of ATP generated per atom of oxygen consumed in the electron transport chain. The P/O ratio als

23、o reflects the ratio of ATPs synthesized per pair of electrons consumed. the P/O ratio was 3(2.5) for NADH oxidation and 2 (1.5)for succinate (that is, FADH2) oxidation,It was widely believed that ATP synthesis occurs by chemical coupling,High-energy Chemical intermediates Phophorylated protein inte

24、rmediates Paul Boyer and the Conformational Coupling Model If the energy of electron transport was not stored in some high-energy intermediate, perhaps it was stored in a high-energy protein conformation.,This model suggested that reversible conformation changes transferred energy from proteins of t

25、he electron transport chain to the enzymes involved in ATP synthesis. This model eventually evolved into the binding change mechanism. Boyers model is supported by a variety of binding experiments and is essentially consistent with Mitchells chemiosmotic hypothesis.,9 The chemiosmotic model was prop

26、osed by Peter Mitchell in 1961 to explain the coupling of electron flow and ATP synthesis,The chemiosmotic model of Mitchell: e- flow and ATP synthesis are separate events, coupled via a transmembrane H+ gradient,protons are driven across the membrane from the matrix to the intermembrane space and c

27、ytosol by the events of electron transport. This mechanism stores the energy of electron transport in an electrochemical potential. As protons are driven out of the matrix, the pH rises and the matrix becomes negatively charged with respect to the cytosol. Proton pumping thus creates a pH gradient a

28、nd an electrical gradient across the inner membrane. Flow of protons which go back into the matrix from the cytoplasm down this electrochemical gradient, an energetically favorable process, then drives the synthesis of ATP.,Oxidative phosphorylation occurs on the inner membrane of mitochondria (cris

29、tae),(or plasma membrane In bacteria),10 ATP Synthase,The mitochondrial complex that carries out ATP synthesis is called ATP synthase or sometimes F1F0ATPase. ATP Synthase Consists of Two ComplexesF1 and F0,ATP synthase was first identified by dissociation and reconstitution studies,Abundant knob-li

30、ke protruding structures were observed on the matrix side of the inner mitochondrial membrane by EM (Racker in 1960).,ATP synthase comprises a proton channel (Fo) and an ATPase (F1),F1: a33.,Fo: ab2c10-12,11 Inhibitors of Oxidative Phosphorylation,Rotenone(鱼藤酮) Ptericidin(杀粉蝶菌素) Amytal(安密妥) Mercuria

31、ls Demerol(德美罗,度冷丁),Uncouplers: 2,4-dinitrophenol Dicumarol(双香豆素) FCCP,Thenoyltri-fluoroacetone Carboxin(噻吩甲酰三氟丙酮),Cyanide Azide Carbon monoxide,Antimycin (抗霉素A),oligomycin(寡霉素),12 Oligomycin and DCCD Are ATP Synthase Inhibitors,The c subunit of F0 is labeled with dicyclohexylcarbodiimide (DCCD,二环己基

32、碳二亚胺), proton flow through F0 is blocked and ATP synthase activity is inhibited. Likewise, oligomycin(寡霉素) acts directly on the ATP synthase. By binding to a subunit of F0, oligomycin also blocks the movement of protons through F0.,13 Uncouplers Disrupt the Coupling of Electron Transport and ATP Syn

33、thase,Another important class of reagents affects ATP synthesis, but in a manner that does not involve direct binding to any of the proteins of the electron transport chain or the F1F0ATPase. These agents are known as uncouplers because they disrupt the tight coupling between electron transport and

34、the ATP synthase. Uncouplers act by dissipating the proton gradient across the inner mitochondrial membrane created by the electron transport system.,Typical examples include 2,4-dinitrophenol(二硝基苯酚), dicumarol(双香豆素), and carbonyl cyanide-p- trifluoromethoxyphenyl hydrazone (perhaps better known as

35、fluorocarbonyl-cyanide phenylhydrazone or FCCP).,These compounds share two common features: hydrophobic character and a dissociable proton. As uncouplers, they function by carrying protons across the inner membrane. Their tendency is to acquire protons on the cytosolic surface of the membrane (where

36、 the proton concentration is high) and carry them to the matrix side, thereby destroying the proton gradient that couples electron transport and the ATP synthase.,Endogenous Uncouplers Enable Organisms To Generate Heat,Ironically, certain cold-adapted animals, hibernating animals, and newborn animal

37、s generate large amounts of heat by uncoupling oxidative phosphorylation. Adipose tissue in these organisms contains so many mitochondria that it is called brown adipose tissue for the color imparted by the mitochondria. The inner membrane of brown adipose tissue mitochondria contains an endogenous

38、protein called thermogenin (literally, “heat maker”), or uncoupling protein, that creates a passive proton channel through which protons flow from the cytosol to the matrix.,14 ATP Exits the Mitochondria via an ATPADP Translocase,ATP and ADP molecules do not readily cross biological membranes. Inste

39、ad, these processes are mediated by a single transport system, the ATPADP translocase. This protein tightly couples the exit of ATP with the entry of ADP so that the mitochondrial nucleotide levels remain approximately constant. For each ATP transported out, one ADP is transported into the matrix.,T

40、ransport occurs via a single nucleotide-binding site, which alternately faces the matrix and the cytosol. It binds ATP on the matrix side, reorients to face the cytosol, and exchanges ATP for ADP, with subsequent movement back to the matrix face of the inner membrane.,15 Shuttle Systems Feed the Ele

41、ctrons of Cytosolic NADH into Electron Transport,The Glycerophosphate Shuttle Ensures Efficient Use of Cytosolic NADH The MalateAspartate Shuttle Is Reversible Occurs in liver, kidney and heart and is readily reversible (depending on which side has a higher NADH/NAD+ ratio. NADH are “transported” by the glycerol phosphate shuttle, it yield only 1.5 ATP; if by malateaspartate shuttle, it yield 2.5 ATP.,Glycerol-3-phosphate shuttle system,16 ATP-Producing Pathwa