生物化學(xué)原理課件（英文）：Chapter20 Biological oxidation

1、楊榮武楊榮武 生物化生物化 學(xué)原理學(xué)原理 第二版第二版 Chapter20 Biological oxidation Outline Definition Comparison with Non-biological oxidation Electron transport chain or Respiratory chain Oxidative phosphorylation (OxP) Inhibitors of ETC & OxP Respiratory control and P/O ratio All oxidation reactions occurring in the livi

2、ng things Common features shared by Non-biological Oxidation 1. Reaction nature is loss of electron or gain of oxygen 2. Release the same amount of energy Unique features 1. Mild reaction conditions 2. Many steps 3. Involving a lot of enzymes and coenzymes Biological Oxidation 1.NAD+ and NAD+ -linke

3、d Dehydrogenases- NAD+ is a mobile electron carrier 2.Flavin and Flavin-linked Dehydrogenases 3.CoQ（UQ）-also a mobile electron carrier 4.Iron-sulfur protein 5.Cytochromes-Cyto c is a mobile electron carrier too 6.O2 Components of ETC Reduction of NAD+ to NADH Reduction of FAD to FADH2 Reduction of C

4、oQ to CoQH2 Iron-sulfur proteins Fe 3+ + e-Fe 2+ Absorbance of cytochromes Different heme groups found in different cytochromes Structure of Cytochrome aa3 1.Measuring E0: Direction of electron flow is E01low E01high (energetically favorable) 2.Spectra analysis: carriers closer to oxygen are more ox

5、idized. 3.Using Specific inhibitors & Artificial e-acceptors 4.Detachment & recombination of Respiratory chain and Assay of individual complexes. Determining the order of electron carriers Standard Reaction Potentials of Respiratory Chain & Related Electron Carriers Detachment of Respiratory chain a

6、nd Assay of individual complexes Complex I NADH Coenzyme Q reductase 2 cofactors1 FMN 6-7 Fe-S centers 2e- transferred4 protons pumped Accepts e- from NADHNADH can only participate in 2 e- transfer reactions Complex II Succinate Coenzyme Q oxidoreductase Has FAD and Fe-S cofactors as electron carrie

7、rs, Succinatefumarate FADFADH2 QH2 Q No ATP is generated, but it gets electrons from FADH2 into the ETC. also cytochrome b. Complex III Coenzyme Q- Cytochrome c oxidoreductase Cytochrome bc1 complex Fe-S centre cytochromes Electron carriers Cytochrome b Cytochrome c1 Can only participate in single e

8、- transfers Complex IV Cytochrome c oxidase Four redox centers Cytochrome a Cytochrome a3 A pair of copper atoms: the CuA centre A copper atom: CuB Electron flow in the complex IV Sequence of ETC Electron flow along the ETC Electron flow along the ETC Coupling e- Transport and Oxidative Phosphorylat

9、ion This coupling was a mystery for many years Coupling mechanism 1.Chemical coupling - Many biochemists squandered careers searching for the elusive “high energy intermediate”, but in vain. 2.Conformational coupling 3.The chemiosmotic hypothesis - Peter Mitchell proposed a novel idea - a proton gra

10、dient across the inner membrane could be used to drive ATP synthesis. Mitchell was ridiculed, but eventually won him a Nobel prize. The proton-motive force (pmf) is the sum of a transmembrane proton concentration (pH) gradient and electric potential Oxidative Phosphorylation The coupling of electron

11、 transport and ATP synthesis Proton Motive Force (pmf) Evidences supporting the chemiosmotic hypothesis How does the proton gradient build up across the inner membrane of mitochondria? 1.Q cycle 2.Conformational change molecular steam engine How does a F1/F0 ATPase catalyze synthesis of ATPs? Paul B

12、oyers binding change mechanism won a share of the 1997 Nobel in Chemistry More questions and more answers Evidence #1 in support of the chemiosmotic hypothesis Evidence #2 in support of the chemiosmotic hypothesis Evidence #3 in support of the chemiosmotic hypothesis F1/F0 ATP Synthase Proton diffus

13、ion through the protein drives ATP synthesis! Two parts: F1 and F0 (latter was originally F-o for its inhibition by oligomycin) 1) F1 catalytic subunit, made of 5 polypeptides with stoichiometry . 2) Fo complex of integral membrane proteins that mediates proton transport. Racker & Stoeckenius confir

14、med Mitchells hypothesis using vesicles containing the ATP synthase and bacteriorhodopsin Q cycle can produce the proton gradient Architecture of respiratory complex I The “steam engine” of the cell? The overall architecture of the complex revealed here strongly supports the idea that proton translo

15、cation is driven by long- range conformational changes . It is likely that movements of several helices in the hydrophilic domain (indicated by arrows) produce a piston-like motion of helix HL along the membrane domain. This movement can synchronously tilt the three nearby discontinuous helices, cha

16、nging the conformation of ionizable residues inside the respective proton channels and resulting in the translocation of three protons. The fourth proton per catalytic cycle may be translocated at the interface of the two domains. Thus, complexI appears to resemble a steam engine, where the energy o

17、f electron transfer is used to move a piston, which then drives, instead of wheels, a set of discontinuous helices. Proposed model of proton translocation by complex I ATP Is Synthesized by the Binding Change Mechanism. The mechanism of ATP synthesis by proton-translocating ATP synthase can be conce

18、ptually broken down into three phases: 1. Translocation of protons carried out by F0. 2. Catalysis of formation of the phosphoanhydride bond of ATP carried out by F1. 3. Coupling of the dissipation of the proton gradient with ATP synthesis, which requires interaction of F1 and F0. Proposed by Paul B

19、oyer The Binding Change Mechanism The Binding Change Mechanism Binding change and rotational catalysis Reaction Mechanism of F1F0-ATP Synthase Consists of two complexes, F0, and F1. F0 unit is bound within Membrane F1 is the catalytic Unit and consists of a, b, g, d, e subunits. Proton flow C unit r

20、otates g rotates conformation change ATP synthesized Matrix Side Asp residues in the c subunits pick up a proton from one side of the membrane. They become uncharged and can then contact the lipid membrane. Protons arriving at the far side, dissociate readily because of the low H+ concentration on t

21、he other side of the membrane This drives the rotor in a unique direction Asp residues OO-OOH + H+ Intermembrane space Asp (low pH) membrane The rotor mechanism - coupling proton flow to rotary motion Evidence#1 in support of the binding change Conclusion: ATP is stabilized relative to ADP on the su

22、rface of F1 Expected: H218O ATP ADP + P(16O)3 (18O)3- Singly labeled Found: H218O ATP ADP + P18O43- Totally exchanged Hydrolysis of ATP catalyzed by ATP synthase (reverse reaction) The proton gradient drives the release of ATP from the enzyme surface ATP is stabilized by binding to enzyme. Free ener

23、gy required for its release is provided by proton- motive force. Evidence#2 in support of the binding change The experimental system used to observe the rotation Researchers from Japan anchored molecules of F1- ATPase to a glass slide and - like putting a flag on top of a pole - attached a long, flu

24、orescent filament of actin to the end of the drive shaft. By bathing the enzyme in ATP, the researchers made F1-ATPase break down the energy molecule and watched as it whirled the fluorescent filament around like a propeller. The rotation of the c-ring in E. coli F1F0-ATPase Inhibitors of Oxidative

25、Phosphorylation N Rotenone inhibits Complex I - and helps natives of the Amazon rain forest catch fish! N Amytal N Antimycin A N Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferric form (Fe3+) of a3 N Oligomycin and DCCD are ATP synthase inhibitors ATP-ADP Translocase ATP must be

26、 transported out of the mitochondria ATP out, ADP in - through a translocase ATP movement out is favored because the cytosol is + relative to the - matrix But ATP out and ADP in is net movement of a negative charge out - equivalent to a H+ going in So every ATP transported out costs one H+ One ATP s

27、ynthesis costs about 3 H+ Thus, making and exporting 1 ATP = 4H+ Some Agents that interfere with OxP Inhibitors of Oxidative Phosphorylation Uncouplers and Uncoupling protein Uncoupling e- transport and oxidative phosphorylation L Uncouplers disrupt the tight coupling between electron transport and

28、oxidative phosphorylation by dissipating the proton gradient L Uncouplers are hydrophobic molecules with a dissociable proton L They shuttle back and forth across the membrane, carrying protons to dissipate the gradient L An uncoupling protein () is produced in brown adipose tissue of newborn mammal

29、s and hibernating mammals. This protein of the inner mitochondrial membrane functions as a H+carrier. The uncoupling protein blocks development of a H+ electrochemical gradient, thereby stimulating respiration. DG of respiration is dissipated as heat. Structures of several uncouplers Action of 2,4-d

30、initrophenol Action of UCP Action of Thermogenin UCP & BAT Most adipocytes are white in color and contain a single globule of FAT surrounded by a thin skin of cytoplasm. A minority of brown adipocytes have multiple smaller globules of fat and a more extensive cytoplasm that is rich in mitochondria a

31、nd respiratory pigments. In contrast to white adipose tissue (WAT), brown adipose tissue (BAT) has a very good blood supply. It is very prominent in newborn animals that have high heat losses, and also in cold-adapted and hibernating species. Differentiation of brown adipocytes is promoted by thyrox

32、ine, which raises basal metabolic rate. Noradrenalin stimulates lipolysis in BAT as it does in WAT, releasing FFA. In WAT, FFAs are released into the circulation, but in BAT they are transported in a cyclical manner across the inner mitochondrial membrane, collapsing the pH and potential gradients and