【生化（英文）教案】Chapter 9 ATP synthesis

1、 ATP SYNTHESISDianzheng Zhang, Ph.D. OBJECTIVES:A. Understand how ATP is generated from metabolic fuelsB. Understand how reducing equivalents are generated from foods and energy storesC. Understand how reducing equivalents are coupled to ATP productionD. Describe the components, structural features

2、and functions of the mitochondrial electron transport systemE. Understand how electron transport is coupled to ATP synthesisF. Understand the relationship between ATP-generating and ATP-requiring systems and their controlOUTLINE:ICoupling of energy expenditure to energy productionIIMitochondrial ATP

3、 productionIIIOxidative PhosphorylationIVGeneration of Reducing Equivalents for energy productionVRegulation of oxidative phosphorylationVIUncoupling Phosphorylation from Electron TransportVII Coupling energy requiring (endergonic) with energy releasing (exergonic) reactionsImportant concepts:1. Oxi

4、dation of foods/body stores provides the energy to synthesize ATP, the useful form of energy required to perform biological work.2. Fatty acids are the preferred fuels for energy metabolism, then ketoacids, and finally glucose if neither fatty acids nor ketoacids are available (except for the brain)

5、.3. Reducing equivalents in the forms of NADH and FADH2 are oxidized by the mitochondrial electron transport system to pump protons across the mitochondrial membrane. This proton pump is coupled to oxidative phosphorylation to produce ATP. References:1.Harper, Chapter 12Role of ATP2.Harper, Chapter

6、13Biological Oxidation3.Harper, Chapter 14Respiratory Chain/Oxidative Phosphorylation4.Marks, Chapter 20Generation of ATP from Metabolic Fuels5.Marks*, Chapter 4Generation of ATP from Metabolic Fuels* Marks, Biochemistry, 3rd edition, Williams and Wilkins Board Review SeriesI. ATP SynthesisATP (aden

7、osine 5-triphosphate) has three phosphoryl groups (, , ) etherify to the 5-position of the ribose moiety (Fig 1). The two terminal ester bonds ( and ) are more energy rich. The energy released from hydrolysis of the -ester bond can be used in a wide range of biological system, and therefore ATP is a

8、lso known as the energy currency. Although ATP can been synthesized through substrate level phosphorylation (see Carbohydrate I), oxidative phosphorylation plays more important role in terms of ATP formation. Fig 1. Molecular structure of ATPEnergy can neither be created nor destroyed. We are going

9、to discuss the energy sources; where and how ATP is synthesized; and special attention will be paid to the regulation of these processes.1. Mitochondrion is one of the important organelles in most animal cells (Fig. 2). The numbers of mitochondria in different cells vary greatly (few hundreds to few

10、 thousands), mainly determined by their energy needs. Mature red blood cells do not have mitochondrion.The architecture of a mitochondrion is schematically shown in figure 3. Fig 2. Organelles in a typical animal cell The outer membrane forms the boundary of the organelle The inner membrane is highl

11、y convoluted to form cristae, and contains the mitochondrial matrixThe Intermembrane space is formed between the outer and the inner membrane Due to the high permeability of the outer membrane, the contents in the inter- membrane space are similar to that of the cytosol The inner membrane is imperme

12、able to almost everything: H+, K+, Na+, ATP, ADP, and Phosphate The inner membrane and the matrix is especially rich in proteins/enzymes involved in different reactions, including the electron transport system (ETS) and ATP synthase Fig 3. Architecture of Mitochondrion2. Electron Transport Systems (

13、ETS) The ETS is formed by four protein complexes (namely Complexes I, II, III and IV, respectively) locate on the mitochondrial inner membrane Each Complex contains multiple proteins and their specific prosthetic groups (Table 1) NADH and FADH2 are the electron donors for Complex I and Complex II, r

14、espectively Both Complex I and Complex II pass their electrons to Complex IIIFig 4. Electron transport system (ETS) Complex III passes electrons to Complex IV Complex IV donates its electrons to O2, and H2O is formed from the reduced O2 The net reactions can be expressed as: Chemicals that can inter

15、rupt the electron transport system will affect the electron flow and ATP synthesis. For example, both cyanide and carbon monoxide are extremely toxic because they can bind to the Fe in the heme rings, obstruct electron flow, and ultimately block ATP synthesis. Table 1: Summary of electron transport

16、system 3. Formation of Electrochemical Potential The Complexes in the ETS not only serve as electron transporters, but also as H+ pumps Protons are pumped by the ETS from the matrix to the intermembrane space Different numbers of H+ are pumped by the Complexes during the transport of two electrons (

17、Table 2) Pumping of H+ will lead to the imbalance of H+ concentration across the inner membrane and this imbalance can be expressed as the Electrochemical potential: Electro: because of membrane potential (positive outside, negative inside) Chemical: because it involves proton gradients Proton gradi

18、ent makes matrix negative with respect to inter-membrane space More H+ in inter-membrane space -> more positively charged Electrochemical potential provides the driving force for ATP synthesis from ADP and PiFigure 5: Proton-motive force across the inner mitochondrial membrane, created by pumping

19、 protons from the matrix across the inner membrane. A. Protonmotive force, or electrochemical potential, due to the membrane potential. B. Protonmotive force, or electrochemical potential, due to the concentration gradient. (20.4 Marks) 4. ATP Synthase (Fo/F1-ATPase)ATP synthase (also known as Compl

20、ex V) is another big protein complex located in the inner mitochondrial membrane. It consists two domains: the Fo-domain and F1-domain. The Fo (composed of 12 peptide subunits in human ATP synthase) forms a pore or channel in the membrane through which the H+ returns to the matrix The proton current

21、 mechanically drives the rotation of Fo as well as the -subunit, which will lead to conformational changes in F1 headpiece. These changes lead to three consecutive steps (For detailed experimental evidence, please see the appendix 2): (1) Binding of substrates Pi and ADP (2) Synthesizing ATP(3) Rele

22、asing of the product ATP It has been experimentally proven that 12 protons are needed to rotate the Fo complex for a full circle (360o). Meanwhile, 3 ATP molecules were synthesized. Therefore, to synthesize an ATP molecule, 4 protons are used Figure 6: ATP-synthase. The Fo subunits form a pore or ch

23、annel through the inner mitochondrial membrane, shown in gray. The subunits of the F1 headpiece form three a, b pairs which each have a binding site for ADP and Pi (20.5 Marks).5. Oxidative phosphorylation is the process of two reactions (oxidation and phosphorylation) coupled with each other: Oxida

24、tion: The coenzymes (NADH or FADH2) get oxidized, electrons transferred through ETS and eventually been accepted by O2 Protons were pumped simultaneously to the intermembrane space from the matrix and electrochemical potential was established Phosphorylation: ADP is phosphorylated to ATP by ATP synt

25、hase using the electrochemical potential as driving force Fig 7. ETS, ATP synthase and oxidative phosphorylation6. Transporters in the mitochondrial inner membrane: ATP is synthesized from ADP and phosphate in the mitochondrial matrix, and been used mainly in the cytosol to form ADP and phosphate. H

26、owever, the inner membrane is permeable to none of these molecules. To ensure the continuation of ATP generation and usage, the transport systems on the mitochondrial inner membrane carry ADP and Pi into the matrix and allow the newly synthesized ATP to leave for the cytosol. Adenosine nucleotide tr

27、anslocase is responsible for transport ATP from the matrix to the cytosol; and ADP from the cytosol to the matrix Phosphate translocase is responsible to bring the phosphate back to the matrix from intermembrane space Since Adenosine nucleotide translocase mediates the movement of ATP and ADP in opp

28、osite direction, adenosine nucleotide translocase is called an antiporter Phosphate translocase mediates phosphate and H+ to move to the same direction, it is called a symporterFig 8. The ATP-ADP transport is favored because the matrix is electrically negative relative to the outside. At pH7, Pi is

29、present as both HPO4-2 and H2PO4-. There is no net flow of charge during symport of H2PO4- and H+, but the relatively low proton concentration in the matrix favors the inward movement of H+. Thus the proton-motive force is responsible both for providing the energy for ATP synthesis by ATP synthase (

30、uniporter) and for transporting substrates (ADP and Pi) in and product (ATP) out of the mitochondrial matrix.7. Summary of Oxidative Phosphorylation Synthesis of ATP involves the inter-conversion of energy from (1) oxidation/reduction (chemical energy) to (2) an electro-chemical gradient (potential

31、energy) which (3) drives the phosphorylation of ADP forming the biologically necessary energy currency of a high-energy phosphate bond on ATP Different numbers of H+ were pumped by different ETS (Complex I to Complex IV vs. Complex II to Complex IV). Thus, different numbers of ATP were synthesized c

32、orrespondingly. Fig 9. ETS, ATP synthase and oxidative phosphorylation8. Reducing Equivalents: A reducing equivalent can be defined as 1 hydrogen atom Since each hydrogen atom can be separated to 1 proton and 1 electron, a reducing equivalent can be expressed either as H or H+ + e Reducing equivalen

33、t can be carried by different molecules (NAD+, NADP+, FAD or FMN)NADH and NADPH (derived from vitamin B3)Fig 10: NAD/NADP and NADH/NADPH (derived from vitamin B3) Both NAD+ and NADP+ are derived from vitamin B3 (niacin). They can be synthesized from tryptophan, a process needs vitamin B6. Vitamin B3

34、 deficiency Glossitis (inflammation of the tongue; swelling or color change of the tongue) Severe deficiency leads to pellagra (three Ds: Diarrhea, Dermatitis, Dementia) Hartnup disease (poor absorption of nonpolar amino acids) Reduced tryptophan availability Malignant carcinoid syndrome Increased t

35、ryptophan metabolism (formation of serotonin) INH (isonicotinylhydrazine) users for the treatment of tuberculosis Vitamin B6 deficiency, a side effect of this drug Excess vitamin B3 (in treatment of hyperlipidemia) Facial flushing (due to pharmacological doses for treatment of hyperlipidemia)Fig. 11

36、: FAD and FADH2 (derived from vitamin B2) Both FAD and FMN are derived from vitamin B2 Deficiency of B2 Cheilosis (inflammation of lips, scaling and fissures at the corners of the mouth) Corneal vascularizationII. ETS, ATP synthase and oxidative phosphorylation¨ Under normal physiological condi

37、tions, the reducing equivalents are mainly generated from three biological fuels: © Carbohydrates (glucose and other sugars)© Fats (fatty acids and glycerol back bone)© Proteins (amino acids)¨ During starvation, ketone bodies serve as major substrates for muscle and/or brain tiss

38、ues¨ Ethanol is also energy rich and contributes reducing equivalents for alcohol drinkers¨ The processes of oxidation of foods/stores, generation of reducing equivalent and ATP synthesis are illustrated in Figure 12, and summarized as following.© Conversion to Acetyl CoA© Oxidat

39、ion of Acetyl CoA via TCA Cycle© Generation of reducing equivalents© Synthesis of ATPFigure 12: Fuel oxidation, reducing equivalent generation and ATP synthesis (Fig 4.1 Marks review)¨ Acetyl CoA is the common terminal product of different catabolic pathways¨ Different fuels have

40、 different efficiencies in generating acetyl CoA (Figure 13)Figure 13: Origin of the acetyl group in various fuels. Acetyl CoA is derived from the oxidation of fuels. The portions of fatty acids, glucose, ketone bodies, ethanol, amino acid and alanine which are converted to the acetyl group of acety

41、l CoA are shown. (fig.19.2 Marks)III. Coupling of energy expenditure to energy productionA. Energy requiring processes: © Anabolism (synthesis)© Mechanical work (muscle contraction)© Active transportation B. ATP synthesis:© Substrate level phosphorylation (see Carbohydrate metabo

42、lism)© Oxidative phosphorylationC.ATP synthesis and ATP usage are two processes coupled with each otherFigure 14: Utilization of high energy bonds in the cell. 1. Glycolysis converts glucose to intermediates with high-energy phosphate bonds, such as phosphoenolpyruvate, whose P bond is transfer

43、red to ADP to form ATP in substrate level phosphorylation. 2. The energy of the high-energy phosphate bond of P is derived from oxidative phosphorylation. 3. In order to oxidize fatty acids, pyruvate, and other compounds to CO2, these compounds are converted to acetyl CoA, which has a high-energy th

44、ioester bond. 4. The P bond can be transferred from ATP to other nucleotides, such as UDP or AMP, to be used in biosynthetic processes. 5. The energy of the P bond of ATP is converted into a conformational change of Na+/K+-ATPase, which transforms the energy into a Na+ gradient. 6. The relative move

45、ment of actin and myosin filaments utilizes P bond energy of ATP. ATP can be generated directly at the site of utilization from the P bond energy of creatine phosphate. (fig. 18.12 Marks)IV. Reducing equivalent shuttle systemsProblem: NADH cannot cross the inner mitochondrial membrane, so the electr

46、ons and protons must be carried across by the shuttle systems. Solution: two shuttle systems: Malate Aspartate Shuttle and Glycerol-3 Phosphate Shuttle, which carry the reducing equivalent (electrons and protons), but not the NADH molecule, across the mitochondrial membrane.The choice of shuttles de

47、pends on the presence of the appropriate enzymes in that particular cell. The Malate Aspartate Shuttle system is dominant in liver tissues and the Glycerol-3 phosphate shuttle system is dominant in brain tissues. i)Malate/Aspartate Shuttle (mainly in the liver tissues)© NADH firstly reduces oxa

48、loacetate (OAA) to malate catalyzed by malate dehydrogenase (cytosol)© Malate enters the mitochondria through the malate/-ketoglutarate transporter© In the mitochondrial matrix, malate is re-oxidized back to OAA (mitochondrial)© NADH produced by this reaction in the mitochondrion ente

49、rs the electron transport chain reaction, and approximately 2.5 ATPs are generated for each NADH© Because OAA cannot cross the mitochondrial membrane either, it is transaminated to aspartate catalyzed by Glutamate-Oxaloacetate Transaminase (Mitochondrial)© Then aspartate is sent back to th

50、e cytosol via the aspartate-glutamate transporter© In the cytosol, aspartate is de-aminated to regenerate OAA for the cycle to continue (cytosol)© This system share some substrates with the TCA cycleFig. 15: (From: Mathews, Biochemistry, 3rd edition,. Chapters: 9)ii)Glycerol-3-Phosphate Sh

51、uttle (mainly for the brain tissues)© Dihydroxyacetone phosphate (DHAP, produced in the cytosol during glycolysis) is reduced to glycerol 3-phosphate (G3P) by the NADH © Glycerol 3-phosphate is oxidized to DHAP in the inner mitochondrial membrane by the enzyme G3P dehydrogenase that transf

52、ers the electrons to FAD to form FADH2© Then FADH2 pass its electrons to Complex III in the electron transport chain, which generates approximately 1.5 ATP for each FADH2 © DHAP is regenerated and returns to the cytosol for the cycle to continue Fig. 16: (From: Mathews, Biochemistry, 3rd e

53、dition,. Chapters: 9).Note: (1) Different number of ATP molecules will be generated from a cytosolic NADH depending on which shuttle system is used. (2) No mater which shuttle system is used, only the reducing equivalents (electrons and protons), not the NADH itself, are transferred across the mitoc

54、hondrial membrane.V. Regulation of oxidative phosphorylation(a) The role of ADP in respiratory controlExperiment: Isolated mitochondria are incubated in a closed chamber with fixed amount of oxygen in a phosphate buffer. During the oxidative phosphorylation, oxygen is reduced to water. Therefore, th

55、e speed of oxygen reduction (declining of oxygen concentration) represents the speed of oxidative phosphorylation. Figure 17: The rate of oxygen consumption is controlled by the concentration of ADP, or by the phosphate potential (ATP/ADPPi), (Fig. 20.12 Marks). Addition of the substrates, pyruvate

56、and malate, enhances oxidative phosphorylation in certain degree. Addition of ADP speeds up oxygen concentration reduction rapidly When all the ADP been converted to ATP (ADP=0), oxidative phosphorylation slows down dramatically ADP/ATP and NAD/NADH ratios control oxidative phosphorylation: The righ

57、t diagram shows the sequence of events. (1) ADP is phosphorylated to ATP. Therefore, the ratio of ADP/ATP becomes lower. (2) The phosphorylation pulls protons through ATP synthase into the matrix. (3) The use of protons from the cytosolic side for ATP synthesis decreases the proton gradient. (4) As a result, the electron transport chain pumps more protons