1. Chapter 18 Oxidative phosphorylation
Mitochondria, stained green, from a network inside a fibroblast cell. Mitochondria oxidize carbon fuel to form cellular energy in the form of ATP.

2. Outline
18.1 Eukaryotic Oxidative Phosphorylation Takes Place in Mitochondria 18.2 Oxidative phosphorylation Depends on Electron Transfer 18.3 The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a physical Link to the Citric Acid Cycle 18.4 A Protein Gradient Powers the Synthesis of ATP 18.5 Many Shuttles Allow Movement Across the Mitochondrial Membrane 18.6 The Regulation of Cellular Respiration Is Governed Primarily by the Need for ATP

3. Cellular respiration proton-motive force
7okg male  need 8400kJ/day need 83kg ATP
recycle ATP about 300 times/day
Electron-transport chain (respiratory chain): electron flow
NADH + ½ O2 + H+  H2O + NAD+
The proton pumps from mitochondria matrix into the intermembrane space
pH gradient
proton-motive force
TCA
CO2
Cellular respiration
ADP + Pi + H+  ATP + H2O
Fig 18.1 Overview of oxidative phosphorylation

4. 18.1 Eukaryotic Oxidative Phosphorylation Takes Place in Mitochondria
Mitochondria are bounded by a double membrane
Oval shaped, about 2μm in length an 0.5 μm in diameter
Two membrane system:
Outer membrane
Quite permeable to most small molecules and ions
Contains mitochondrial porin (30-35kd pore-forming protein; VDAC; voltage dependent anion channel)
Regulate flux of metabolites, usually anions, such as phosphate, chloride, organic anions, and adenine nucleotides
Highly folded inner membrane -- cristae
Intermembrane space
Matrix
(A)
(B)
Fig 18.2 Electron micrograph (A) and diagram (B) of a mitochondria.

5. Mitochondria are bounded by a double membrane
Oval shaped, about 2μm in length an 0.5 μm in diameter
Two membrane system:
Outer membrane
Highly folded inner membrane -- cristae
Human contain an estimated 14,000m2 of inner membrane
Oxidative phosphorylation
Impermeable to nearly all ions and polar molecules
A large family of transporters shuttles metabolites such as ATP, pyruvate, and citrate across the inner membrane
The two faces of this membrane
Matrix side (N sides): membrane potential is negative
Cytoplasmic side (P sides): membrane potential is positive
Intermembrane space
Matrix
TCA cycle
Fatty acid oxidation

6. Biochemical anatomy of a mitochondrion
(MW<5000)
一個肝臟粒線體的內膜至少有超過 10,000 電子傳遞系統 (electron-transfer systems)及 ATP 合成酶 synthase
而心臟的粒線體具較多的cristae並超過3倍肝臟內膜的區域，具有更多的電子傳遞系統
無脊椎動物、植物及微小脊椎動物的粒線體與脊椎動物相似，惟其大小、形狀及內膜內凹程度各有不同
specific transporters

7. In prokaryotes
The electron-driven proton pumps and ATP-synthesizing complex are located in the cytoplasmic membrane, the inner of two membranes
Outer membrane of bacteria is permeable to most small metabolites (porins)
Proton gradient +
Membrane potential
電子傳遞導致質子被送出穿越粒線體內膜
Fig 20.1 A proton gradient is established across the inner mitochondrial membrane as a result of electron transport

8. Mitochondria are the result of an endosymbiotic 內共生 event
Mitochondria are semiautomous organelles –endosymbiotic relation with the host cell
Contain DNA
Encodes a variety of different proteins and RNAs
Circular or linear form DNA
The genomes range broadly in size across species
Human mitochondria DNA 16569bp, encodes 13 respiratory-chain proteins, rRNAs, tRNAs
Fig 18.3 sizes of mitochondria genomes.

9. Universal Electron Acceptors
氧化磷酸化反應起源於電子進入呼吸鏈中
靠的是去氫酶(dehydrogenase)的作用
主要的電子接受器(electron acceptor) 為nicotinamide nucleotide (NAD+ or NADP +)或flavin nucleotide (FMN or FAD)
nicotinamide nucleotide-linked dehydrogenase
Reduced substrate + NAD+ 
Oxidized substrate + NADH + H+
Reduced substrate + NADP+ 
Oxidized substrate + NADPH + H+
每次移走兩個氫原子
Flavoproteins
可接受1或2個電子 (semiquinone form or FADH2, FMNH2)

11. 18.2 Oxidative phosphorylation Depends on Electron Transfer
In oxidative phosphorylation, the electron-transfer potential of NADH or FADH2 is converted into phosphoryl-transfer potential of ATP
A useful way to look at electron transport is to consider the change in free energy associated with the movement of electrons from one carrier to another (自由能的改變與電子的移動)
reduction potential (oxidation-reduction potiential)
A carrier of high reduction potential will tend to be reduced if it is paired with a carrier of lower reduction potential (較高還原電位者被還原;較高還原電位者會得到電子)

12. Ethanol acetaldehyde + 2H++ 2e- Fumarate + 2H++ 2e- succinate
依電流方向得知還原電位的高低
Ethanol acetaldehyde + 2H++ 2e-
Eo’=-0.197V
Fumarate + 2H++ 2e- succinate
Eo’=+0.031V
較高還原電位
Fig 20.3 Experimental apparatus used to measure the standard reduction potential of the indicated redox couples: (a) the ethanol/acetaldehyde couple; (b) the fumarate/succinate couple

13. Standard biological voltage
Base on
1M, pH 7 at 25oC
(standard state)

15. NAD+ + 2H+ +2e-  NADH + H+ Eo’=-0
NAD+ + 2H+ +2e-  NADH + H+ Eo’=-0.320V During redox reaction: NADH + H+  NAD+ + 2H+ +2e- Eo’=0.320V ½ O2 + 2H+ +2e-  H2O Eo’=0.816V NADH + ½ O2 + H+  NAD+ + H2O Eo’ =1.136V
Go: free energy change in the standard state
n: the number mole of electron transferred
F: Faraday’s constant (96.485kJV-1mol-1)
Eo’: the voltage for the two half reactions
When Eo’ is positive Go is negative
Go=-nFEo’
Go=-(2)(96.485kJV-1mol-1)(1.136V)
= -219kJ mol-1
NADH passes its electrons along a chain that eventually lead to oxygen, but it does not reduce oxygen directly

16. 18.3 The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a physical Link to the Citric Acid Cycle
Four separate respiratory complexes can be isolated from the inner mitochondria membrane –by fractionation 在粒線體內膜可分離出四種呼吸複合體
Multienzyme systems 屬多酵素的系統 (super molecule complex termed the respirasome)
Complex I: NADH-Q oxidoreductase 氧化還原酶
Complex II: succinate-Q reductase
Does not pump protons
Complex III: Q-cytochrome c oxidoreductase
Complex IV: cytochrome c oxidase 氧化酶
Each of the respiratory complexes can carry out the reactions of a portion of the electron transport chain 每個呼吸複合體都可完成電子傳遞鏈中一部份的反應

18. Fig 18.6 Components of the electron-transport chain

19. Fig 20-6 The electron transport chain, showing the respiratory complexes

20. Two special electron carriers ferry the electrons from complex to the next
Coenzyme Q (Q) : ubiquinone (泛醌)
Hydrophobic quinone
Diffuses rapidly within the inner membrane
Electrons are carried from NADH-Q oxireductase to Q-cytochrome c oxidoreductase
FADH2 generated by the TCA cycle are transferred to ubiquinone to the Q-cytochrome
With a long tail consisting of five carbon isoprene units (contain 10 units  coenzyme Q10)
Three oxidation states: Q, QH., QH2
Electron-transfer reactions are coupled to proton binding and release
Cytochrome C

21. Fig 18.7 Oxidation states of quinones

22. Two special electron carriers ferry the electrons from complex to the next
Coenzyme Q (Q) : ubiquinone (泛醌)
Cytochrome C
Small soluble protein
Shuttles electrons from Q-cytochrome c oxidoreductase to cytochrome c oxidase
Catalyzes the reduction of O2

23. The high potential electrons of NADH enter the respiratory chain at NADH-Q oxidoreductase
Complex I: NADH-CoQ oxidoreductase 氧化還原酶
Catalyzes the first steps of electron transport
Transfer of electrons from NADH to coenzyme Q (CoQ)
L-shaped, with horizontal arm lying in the membrane and a vertical arm projects into the matrix
An enormous enzyme consisting ~46 polypeptides
Proteins contain an iron-sulfur clusters
Flavoprotein that oxidizes NADH
Has a flavin coenzyme  flavin mononucleotide (FMN)
Fig 18.8 Oxidation states of flavins

24. Fe-S clusters in iron-sulfur proteins (nonheme iron proteins)
NADH-Q oxidoreductase contain both 2Fe-2S and 4Fe-4S clusters
Iron ions in these Fe-S complexes cycle between Fe2+ (reduced) and Fe3+ (oxidized) state.
Undergo oxidation-reduction reactions without releasing or binding protons.
2Fe-2S cluster contains 2 irons, 2 sulfides and 4 cys.
Single iron ion bound by 4 cys.
Fig 18.9 Iron-sulfur clusters
4Fe-4S cluster contains 4 irons, 4 sulfide ions and 4 cys.

25. NADH + H+ + E-FMN  NAD+ + E-FMNH2
All the redox reactions take place in the extramembranous part of the enzyme
First step
Electrons are passed from NADH to FMN
NADH + H+ + E-FMN  NAD+ + E-FMNH2
E-FMN: the flavin is covalently bonded to the enzyme
Second step
The reduced flavoprotein is reoxidized
The oxidized form of the iron-sulfur protein is reduced
E-FMNH2 + 2Fe-Soxidized  E-FMN + 2Fe-Sreduced + 2H+
Third step
The reduced iron-sulfur protein then donates its electron to coenzyme Q (CoQ; ubiquinone 泛醌)
Reduced to CoQH2 (還原成CoQH2)
2Fe-Sreduced + CoQ +2H+  2Fe-Soxidized + CoQH2
Over all equation 反應總方程式
NADH + 5H+matrix + CoQ  NAD + + CoQ H2 +4H+cytoplasm

26. Pump 4 hydrogen ions out of the matrix
Q takes up 2 protons and 2 electrons from the matrix and reduced to QH2
Fig Coupled electron-proton transfer reactions through NADH-Q oxidoreductase

27. Ubiquinol is the entry point for electrons from FADH2 of plavoproteins
FADH2 enter the electron-transport chain at the complex II (succinate-Q reductase complex)
Integral membrane protein of the inner membrane
FADH2 electrons are transferred to Fe-S centers
Then finally to Q to form QH2
Does not pump protons
Less ATP is formed from the oxidation of FADH2 than from NADH
Two other enzymes, glycerol phosphate dehydrogenase and fatty acyl CoA dehydrogenase transfer high potential electrons from FADH2 to Q to form ubiquinol
Also do not pump protons

28. Electrons flow from ubiquinol to cytochrome c through Q-cytochrome c oxidoreductase
Electrons from QH2 to cytochrome c by complex III (Q-cytochrome c oxidoreductase; cytochrome reductase)
The overall reaction:
QH2 + 2cyt cox+ 2H+matrix  Q + 2cyt cred + 4H+cytoplasm
The components of Q- cytochrome c oxidoreductase
Three hemes (iron-protoporphyrin IX)
Cytochrome b (Cytochrome bH and bL (low for low affinity))
Cytochrome c1
The iron ions of a cytochrome alternates between a reduced ferrous (+2) state and a oxidized ferric (+3)state
Several iron-sulfur proteins – 2Fe-2S center (Rieske center)
One of the irons ions is coordinated by two histidine residues
Stabilized the center
Cytochromes can carry electrons, but no hydrogens

29. Fig 18.11 Structure of Q-cytochrome c oxidoreductase (cytochrome bc1)

30. The Q cycle funnels electrons from a two-electron carrier to a one-electron carrier and pumps protons
QH2 passes two electrons to complex III, but the electrons acceptor cytochrome c can accept one electron
Q cycle
Two QH2 molecules bind to the complex consecutively, each giving up two electrons and two H+
Q0: first Q binding site
Qi: second Q binding site
Complex III
Complex III
Fig Q-cycle

31. 整個反應釋出4的氫離子
FIGURE The Q cycle, shown in two stages.

32. Q cycle Over all equation
One electron is passed from reduced CoQ to the iron-sulfur clusters to cytochrome c1  semiquinone
CoQH2 + Cyt c1 (oxidized) 
Cyt c1 (reduced) + CoQ- +2H+
Semiquinone in a cycle process  two b cytochromes are reduced and oxidized
A second molecule of CoQ transfer a second electron to cytochrome c1 and to the cytochrome c
electrons to be transferred one at a time from coenzyme Q to cytochrome C 由CoQ每次傳送一個電子到細胞色素c1
Over all equation
2QH2 + Q + 2 cyt cox + 2H+matrix  QH2 +2 Q + 2 cyt cred + 4H+cytoplasm

33. Cytochrome c oxidase catalyzes the reduction of molecular oxygen to water
Complex IV: cytochrome c oxidase 細胞色素c氧化酶
The transfer of electrons from cytochrome c to oxygen
4cyt c [Fe(II)] + 8H+matrix + O2  4cyt c [Fe(III)] + 2H2O + 4H+cytoplasm
The components of this complex
Integral protein of the inner mitochondrial membrane
Three Cu2+ (Two copper center; CuA/CuA and CuB) Cu+  Cu2+
Two heme A groups
Formyl group replaces methyl groups
C17 replaces vinyl group
Not covalently attached protein
heme a, heme a3

34. O22- Fig 18.15 peroxide bridge Electrons stop at CuB and heme a3
Reduced state
Oxygen binding
4cyt c [Fe(II)] + 8H+matrix + O2  4cyt c [Fe(III)] + 2H2O + 4H+cytoplasm
Fig cytochrome c oxidase mechanism

35. FADH2
Fig The electron-transport chain

36. Toxic derivatives of molecular oxygen such as a superoxide radical are scavenged by protect enzyme
Reactive oxygen species (ROS)
Superoxide
ion
Peroxide
the transfer of a single electron to O2 forms superoxide anion, where the transfer of two electrons yields peroxide.
cytochrome c oxidase hold O2 tightly between Fe and Cu ions.
superoxide dismutase
A Mg-containg enzyme located in mitochondria
A Cu- and Zn-dependent cytoplasmic form
Superoxide
dismutase
O2- .
+ 2H+ O2
+ H2O2
catalase
2 H2O2 O2
+ 2H2O

37. Glutathione peroxidase also plays a role in scavenging H2O2
Antioxidant vitamins: vitamins E and C
Vitamine E (lipophilic) is especially useful in protecting membranes from lipid peroxidation
Fig superoxide dismutase mechanism

38. 動脈粥狀硬化
肺氣腫 ; 支氣管炎
裘馨氏肌肉失養症
唐氏綜合症

39. 18.4 A Protein Gradient Powers the Synthesis of ATP
In electron transport
NADH + ½ O2 + H+  H2O + NAD +
ΔGo’ = kJmol-1
How this process is coupled to the synthesis of ATP
ADP + Pi +H+  ATP + H2O
ΔGo’ =+30.5 kJmol-1
Mitochondrial ATPase or F1F0 ATPase or Complex V

40. The Mechanism of Coupling in Oxidative phosphorylation
Membrane potential 0.14V
Fig chemiosmotic hypothesis
In 1961, Peter Mitchell: The chemiosmotic hypothesis
Electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane
Electron transfer through the respiratory chain
pumping of protons from the matrix to the cytoplasmic side of the inner mitochondrial membrane
[H+] is lower in the matrix
Protons them flow back into the matrix
Drive the synthesis of ATP by ATP synthase
The energy-rich unequal distribution of protons is called the proton-motive force power the synthesis of ATP
Chemical gradient: pH gradient
Charge gradient

41. Proton-motive force (Δp) = chemical gradient (ΔpH) + charge gradient (Δψ)
The respiratory chain and ATP synthase are biochemically separate systems, linked only by a proton-motive force
NADH oxidation is coupled to ATP synthesis are:
Electron transport generates a proton-motive force
ATP synthesis by ATP synthase can be powered by a proton-motive force
How??
Fig Testing the Chemiosmotic hypothesis

42. ATP synthase is composed of a proton-conducting unit and a catalytic unit
Large, complex enzyme
Two components:
F0 subunits
Hydrophobic segment that span in the inner mitochondrial membrane
Contains the proton channel of the complex
Consists of a ring comprising from c subunits that are embedded in the membrane
A single a subunit binds to the outside of the ring
F1 subunits
85-Å-diameter, protrudes into the matrix
Catalytic activity of the synthase
Consists of 5 types of polypeptide chains (α3, β3, γ, δ, ε)
The α and β subunits arranged in a hexameric ring
β subunits participate directly in catalysis
γ subunit with a long helical coiled coil that extends into the α3β3 hexamer (make β subunits unequivalent)
F0 and F1 are connected by the central γε unit and by an exterior column (a, 2b and δ)
Fig Structure of ATP synthase

43. Proton flow through ATP synthase leads to the release of tightly bound ATP: the binding-change mechanism
Fig ATP-synthesis mechanism
ADP and ATP complex with Mg2+
Isotopic exchange experiment:
Enzyme-bound ATP forms readily in the absence of a proton-motive force
The role of the proton gradient is not to form ATP but to release it from the synthase
Fig ATP forms without a proton-motive force but is not released

44. Three β subunits are components of the F1 moiety of the ATPase
Three active sites on the enzyme
Each performs different functions
The proton-motive force causes the three active sites to sequentially change functions
The enzyme consists:
The moving unit – rotor
c ring and the γε stalk
The stationary unit – stator
Remainder of the molecule

45. Binding-change Mechanism for proton-driven ATP synthesis
A β subunit performs each of three sequential steps in the synthesis of ATP by change conformation
ADP and Pi binding
ATP synthesis
ATP release
Loose conformation (L)
Binds ADP and Pi
Tight conformation (T)
Binds ATP with great avidity
catalytically active
Convert bound ADP and Pi into ATP
Open form (O)
low affinity for substrate
Bind or release adenine nucleotide
Fig ATP synthase nucleotide- binding site are not equivalent

46. The rotation of the γ subunit drives the interconversion of these three forms
ATP can be synthesis and released by driving the rotation of the γ subunit
Fig Binding -change mechanism for ATP synthase

47. How does proton flow through F0 drive the rotation of the γ subunit?
Dependent on the a and c subunits of F0
Stationary a subunits directly abuts membrane-spanning ring formed by c subunits
Subunit a includes two hydrophilic half-channels
Directly interacts with one c subunit
Protons can enter and pass partway but not complete through the membrane
c subunits
A pair of α helix (span the membrane)
An aspartic acid in the middle
Allow protons to enter
Fig components of the proton-conducting unit of ATP synthesis

48. In proton rich environment
A proton will enter a channel (cytoplasmic half channel)
Bind the aspartate residue of c subunits
Fig proton path through the membrane
(c subunit)
Fig proton motion across the membrane drives rotation of the c ring

49. How does the rotation of the c ring lead to the synthesis of ATP?
The c subunit is tightly linked to the γ and ε subunits
As the c ring turn
The γ and ε subunits are turned inside the α3β3 hexamer of F1
Each 360-degree rotation of the γ subunit
The synthesis and release of three molecules of ATP
If there 10 c subunits in the ring
Transfer 10 protons
Each ATP generated requires 10/ 3 = 3.3 protons
The true value may differ
NADH pump enough protons to generate 2.5 molecules ATP; FADH2 yield 1.5 molecules of ATP

50. The inner mitochondrial membrane is impermeable to protons
FIGURE Chemiosmotic model.
The inner mitochondrial membrane is impermeable to protons
protons can reenter the matrix only through proton-specific channels (Fo).
The proton-motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the F1 complex associated with Fo.

51. 18.5 Many Shuttles Allow Movement Across the Mitochondrial Membrane
Electrons from cytoplasmic NADH enter mitochondria by shuttles
The inner mitochondrial membrane is impermeable to NADH and NAD+
Using
Glycerol 3-phosphate shuttle
Malate-aspartate shuttle

52. Glycerol 3-phosphate shuttle
Transfer a pair of electrons from NADH to dihydroxyacetone phosphate to form glycerol 3-phosphate
From glycolysis
(move into the mitochondria)
(membrane bound isozyme)
Outer surface of the inner mitochondrial membrane
Cytoplasmic NADH transported by glycerol 3-phosphate shuttle
 1.5 molecules of ATP are formed
especially in muscle to sustain high rate of oxidative phosphorylation

53. Malate-aspartate shuttle
In heart and liver
From glycolysis
Malate dehydrogenase
Aspartate aminotransferase
Cytoplasmic NADH transported by malate-aspartate shuttle
 2.5 molecules of ATP are formed

54. The entry of ADP into mitochondria is coupled to the exit of ATP by ATP-ADP translocase
ATP and ADP do not diffuse free across the inner mitochondrial membrane
Specific transport protein, ATP-ADP translocase
Highly abundant (15% protein of the inner membrane)
30kd translocase contains a single nucleotide-binding site
Face the matrix and the cytoplasmic side of the membrane
ADP enters the mitochondrial matrix only if ATP exits
ADP3-cytoplasm + ATP4-matrix  ADP3-matrix + ATP4-cytoplasm
ADP and ATP bind to the translocase wothout Mg2+
ATP transport out of the matrix and ADP transport into the matrix
ATP has one more negative charge
Membrane potential

55. Fig 18.36 Mechanism of mitochondrial ATP-ADP translocase

56. More than 40 such carriers are encoded in the human genome
Synthasome: ATP-ADP translocase + phosphate carrier + ATP synthase
Contain 3 tandem repeats of a 100 a.a. module that come together to form a binding site
Fig mitochondrial transporters
More than 40 such carriers are encoded in the human genome

57. 18.6 The Regulation of Cellular Respiration Is Governed Primarily by the Need for ATP

58. The rate of oxidative phosphorylation id determined by the need for ATP
When ADP concentration rises, the rate of oxidative phosphorylation increase to meet the ATP needs
Respiratory control or acceptor control
Electron do not flow from fuel molecules to O2 unless ATP needs to by synthesis

59. Regulated uncoupling leads to the generation of heat
Some organisms possess the ability to uncouple oxidative phosphorylation from ATP synthesis to generate heat
Maintain body temperature in hibernating animals, in some newborn animals, and adult mammals
The uncoupling is in brown adipose tissue (BAT)
Specialized tissue for the process of nonshivering thermogenesis (指為因應環境溫度改變，增加能量消耗的產熱反應; 非顫抖產熱反應)
Very rich in mitochondria called brown fat mitochondria
The combination of the greenished-colored cytochromed in the mitochondria and the red hemoglobin
Mitochondria contains a large amount of uncoupling protein (UCP-1) or thermogenin (a dimer of 33kd that resemble ATP-ADP translocase
褐色脂肪組織於新鮮活體狀態呈褐色，其脂肪細胞內儲存多量之小型油滴，細胞核呈橢圓形且細胞質內富含粒腺體。褐色脂肪細胞在脂紡氧化之過程，不形成 ATP 而將所 含之能量以熱能形式釋放，提供動物體溫與熱能之需求，在小型哺乳動物之體溫維持上 與動物冬眠之甦醒時，扮演極重要之角色。人類之褐色脂肪組織從出生後到生命之前十 年內會逐漸減少，而白色脂肪組織則相反，所以成為成人之脂肪組織。

60. Fig 18.38 Action of an uncoupling protein

61. Oxidative phosphorylation can be inhibited at many stages
Inhibition of the electron-transport chain
Block the transfer of electrons from the flavoprotein NADH reductase to coenzyme Q
Barbiturates巴比妥鹽藥物
Amytal
Rotenone
The blockage occur involving the electron transfer of b cytochromes, coenzyme Q, and cytochrome c1
Antimycin A抗黴素A
myxothiazol
UHDBT
The blockage occur involving the electron transfer from cytochromes aa3 complex to oxygen
Cyanide (CN-)
Azide (N3-)
Carbon monoxide (CO)

62. 2. Inhibition of ATP synthase
Oligomycin寡黴素
Antifungal agent
Prevent the influx of protons through ATP synthase
3. Uncoupling electron from ATP synthesis
2,4-dinitrophenol (DNP) 2,4-二硝酚
Electron transport from NADH2 proceeds in a normal fashion
ATP is not form by mitochondrial membrane
4. Inhibition of ATP export
Atractyloside
Binds to the translocase when its nucleotide site face the cytoplasm
bongkrekic acid
Binds when this site faces the matrix