1
Q
Convergence at oxidative phosphorylation
A
All of the steps in the degradation of carbohydrates, fats and amino acids converge at oxidative phosphorylation
2
Q
Oxidative Phosphorylation Implications
A
The energy of oxidation indirectly drives the synthesis of ATP
3
Q
Photosynthetic Organism energy capture
A
Capture energy of sunlight and harness it to make ATO in photophorsphorylation
4
Q
Oxidative Phosphorylation Definition
A
Reduction of O2 to H2O
5
Q
Photophosphorylation Definition
A
Oxidation of H2O to O2
6
Q
Similarities between oxidative phosphorylation and photophosphorylation
A
- Electron flow through a chain of membrane-bound carriers
- Free energy of exergonic “downhill” electron flow is coupled to transport of protons “uphill” across a proton-impermeable membrane
- Transmembrane flow of protons down their concentration gradient through specific protein channels provides free-energy to drive ATP synthesis
7
Q
Oxidative phosphorylation in mitochondria
A
- Outer membrane permeability
- Inner membrane permeability
8
Q
Outer mitochondrial membrane permeability
A
Permeable to small molecules (
9
Q
Inner mitochondrial membrane permeability
A
Contains membrane-bound carriers of electrons as well as ATP synthase to make ATP and transporters including translocases which transport ATP out of the mitochondria
10
Q
Electron acceptors for oxidations
A
- Nicotinamide nulceotide-linked dehydrogenases
- Flavoproteins
11
Q
Nicotinamide nucleotide-linked dehydrogenases
A
Enzymes that pass electrons during the oxidation of a substrate to FMN or FAD
12
Q
FMN and FAD electron acceptors
A
- Each can accept one (semiquinone) or two electrons (FMNH2/FADH2)
- Two electrons are tightly bound, sometimes covalently
13
Q
Membrane-bound carriers
A
- Ubiquinone/Coenzyme Q
- Cytochromes
- Iron-sulfur proteins
14
Q
Ubiquinone/Coenzyme Q General Information
A
Lipid-soluble benzoquinone head group with long isoprenoid lipid chain tail
15
Q
Cytochromes General Information
A
Proteins with an iron-containing heme prosthetic group
16
Q
Iron-sulfur proteins General Information
A
Proteins containing iron associated with inorganic sulfur atoms or the sulfur atoms of Cys or both
17
Q
Uboquinone/Coenzyme Q
A
- Lipid soluble benzoquinone with a long isoprenoid tail
- Allows free diffusion within the lipid bilayer of the inner mitochondrial membrane
- Can accept one electron and one proton to form a semiquinone
- Can accept electrons and two protons to form a ubiquinol
- Only accepts one at a time!
18
Q
Cytochromes
A
- Inner mitochondrial membrane bound (except cytochrome c)
- Have associated or covalently bonded iron-containing heme prosthetic groups
- Fe3+ in the heme can directly accept an electron forming Fe2+
19
Q
Iron-sulfur proteins
A
- Contain iron-sulfur centers
- Irons are present as Fe3+, Fe2+ or sometimes there are multiple irons with different charges
- All centers participate in one electron transfers, oxidizing or reducing
- Has 1, 2 or 4 irons
20
Q
Iron-sulfur centers
A
One or more irons are coordinated to the sulfur residues and possible inorganic sulfur atoms
21
Q
Four isolatable multi enzyme complexes in electron transport
A
-Each can be physically separated and individually catalyze electron transfer through a portion of the chain
22
Q
Complex 1 Name and Prosthetic Groups
A
- NADH Dehydrogenase
- Prosthetic Groups: FMN, Fe-S
23
Q
Complex 2 Name and Prosthetic Groups
A
- Succinate Dehydrogenase
- Prosthetic Groups: FAD, Fe-S
24
Q
Complex 3 Name and Prosthetic Groups
A
- Ubiquinone cytochrome c oxidoreductase
- Prosthetic Groups: Heme, Fe-S
25
Cytochrome C Name and Prosthetic Groups
- Not part of a complex
| - Prosthetic Groups: Heme
26
Complex 4 Name and Prosthetic Groups
- Cytochrome Oxidase
| - Prosthetic Groups: Heme, CuA, CuB
27
NADH's path through the electron transport chain
Complex 1 -> Coenzyme Q -> Complex 3 -> Cytochrome C -> Complex 4 -> H2O
28
FADH2 (succinate)'s path through the electron transport chain
Complex 2 -> Coenzyme Q -> Complex 3 -> Cytochrome C -> Complex 4 -> H2O
29
Complex 1
- NADH to Ubiquinone
| - The energy from electron transfer drives the proton pump
30
Complex 1 Catalysis
1. Exergonic transfer of a hydride ion from NADH and a proton from the matrix to ubiquinone
2. Endergonic transfer of four protons from the matrix to the inter membrane space
31
Complex 2
- Succinate to Ubiquinone
- Catalyze oxidation of succinate to fumarate passing electrons to the covalently bonded FAD
- Heme b site may prevent leaking of O2
32
Movement of electrons from Complex 2 from FAD
-Electrons are then passed through the three 2Fe-2S centers to ubiquinone
33
Flavoproteins to Ubiquinone
- Glycerol 3-phosphate dehydrogenase
| - Acyl-CoA dehydrogenase
34
Glycerol 3-phosphate dehydrogenase
-Bypasses complexes 1 and 2 and passes electrons directly from FADH2 to ubiquinone
35
Acyl-CoA dehydrogenase
-Passes electrons to electron transferring flavoprotein then to EFT: ubiquinone oxidoreductase then finally to ubiquinone
36
Complex 3
- Ubiquinone to Cytochrome c
- couples the transfer of electrons from ubiquinol to chyotchrome c with transport of four protons from the matrix to the intermembrane space
37
Electron transfer in complex 3
electron transfer occurs from ubiquinone through a 2Fe-2S center and 3 different cytochromes (2 b type and 1 c type) finally to cytochrome c
38
Complex 4
- cytochrome c to O2
- couples the transfer of 4 electrons from cytochrome c to molecular oxygen reducing it to H2O with th epumping of 4 protons from the matrix to the intermembrane space
39
Electron transfer in complex 4
electrons are transferred from cytochrome c -> binuclear copper center -> coordinated to 2 -SH of Cys -> CuA -> heme a -> heme a3 -> CuB -> O2
40
Electron transfer energy conservation
- Energy is conserved in the gradient
- The standard free energy is very favorable thermodynamically
- The actual free energy is even more thermodynamically favorable as the NAD/NADH is below unity
41
Proton transfer by complexes
4 by Complex 1
4 by Complex 3
2 by Complex 4
42
Proton Motive Force
Energy stored in the gradient
43
Chemical Potential Energy
Difference in concentration in two regions separated by a membrane
44
Electrical Potential Enrergy
Difference in charge across a membrane
45
pH difference across the proton gradient
0.75
46
Free energy for pumping protons outward
- found using pH
- 20 kJ/mol
- pumping 10 moles of protons would give 200 kJ/mol
- 200 kJ/mol of the 220 released by the oxidation of a moles of NADH is conserved in the proton gradient
- about a 10% loss
47
ATP synthesis thermodynamics
- electron transfer of a mole of electron pair from NADH results in about 200 kJ/mol of free-energy
- there is more than enough free energy from this transfer of electrons to synthesize a mole of ATP
48
Chemiosmotic model
-electrochemical energy due to the difference in proton concentratio/change across the membrane drives the synthesis of ATP as protons flow back into the matrix through a proton pore in the protein ATP synthase
49
Chemiosmotic
describes the enzymatic reactions that involve coupling a chemical reaction and a transport process
50
Isolated mitochondria
- with ADP, Pi, and substrate in solution
- causes;
1. substrate oxidation
2. O2 comsumption
3. ATP synthesis
51
Inhibition of electron transport
ultimately leads to O2 blocking ATP synthesis
52
Inhibition of ATP synthesis
- blocks electron transfer in intact mitochondria
| - 2,4-dinitrophenol
53
2,4-dinitrophenyol
- carries protons across the membrane, dissipating the proton gradient and uncoupling the two processes
- allows electron transport without ATP synthesis
54
FoF1 ATPase/ATP synthase
-found on the inner mitochondrial membran in animal cells
55
ATP synthase structure
- FoF1 has two distinct associated components
| - F1 and Fo
56
F1 Structure
-a peripheral membrane protein containing the active site for ATP hydrolysis/synthesis
-3 α and 3 β alternating subunits arranged like segments of an orange
-3 additional subunits γ (central shaft), δ,
& ε
-ATP/ADP binds on the β subunit.
57
Fo Structure
-an integral membrane protein containing the proton pore
-Fo is composed of three distinct
subunits in the proportion ab2c10-12.
-Subunit c is composed of 2
transmembrane helices and
associated to form a cylindrical pore.
58
F1 stabilization
- has a higher affinity for ATP over ADP
- 40 kJ more binding energy for ATP
- the binding energy of the product, ATP, on the enzyme surface lowers the activation energy facilitating formation of the product
59
Release of ATP
the release of ATP from ATP synthase is a major energy barrier rather than the formation of ATP
60
ATP synthase substrate/product binding
- the conformations of the 3 β are different
- when crystallized with ADP each of the subunits are different
- one with ATP
- one with ADP
- one empty
61
ATP synthase conformational changes
-γ moves causing a conformational change to force ATP out of the active site
62
Car analogy
-ATP synthase is like putting a car on the top of a hill and pushing it down and having it produce gas
63
ATP synthase mechanism
- rotational catalysis
- protons passing through the Fo subunit cause the cylinder of c subunits and the γ subunit of F1 to rotate
- rotation causes each of the β subunits to interact differently, changing their conformation making ATP dislocate
64
Stoichiometry of O2 consumption and ATP synthesis
- 10 electrons are pumped out per NADH
- 6 electrons are pumped out per FADH2
- it takes the passage of 4 electrons to generate one ATP
65
P/O ratios
```
NADH = 10/4 =2.5 ATP
FADH2 = 6/4 = 1.5 ATP
```
66
Adenine nucleotide translocase
- an antiporter
- moves 1 ADP into the matrix and 1 ATP into the intermembrane space
- driven by proton motive force; drives 4 (-) out and 3 (-) into the matrix
- effectively uses/cancels out one proton of the gradient
67
Cytosolic to mitochondrial NADH for oxidation
- NADH dehydrogenase only accepts electrons from NADH in the mitochondria
- special shuttle systems using transporter proteins allow indirect conversion of cytosolic NADH into mitochondrial NADH
68
Shuttle systems
- Malate-aspartate shuttle
| - Glycerol 3-phosphate shuttle
69
Malate-aspartate shuttle
- electrons from NADH are passed into the matrix as malate using the malate-α-ketoglutarate transporter
- malate is converted to aspartate and transported out of the matrix with the glutmate-aspartate transporter
70
Glycerol 3-phosphate shuttle
- cytosolic glycerol 3-phosphate dehydrogenase uses NADH to pass electrons to glycerol 3-phosphate
- mitochondrial glycerol 3-phosphate dehydrogenase uses FAD to pass electrons from glycerol 3-phosphate to ubiquinone
- only produces 6 e- moved
- skip complex 1 and complex 2
71
Why have the glycerol 3-phosphate shuttle?
- primarily used in the brain and skeletal muscle
- used to rapidly regenerate NAD+ so that glucose can be oxidized to provide more energy
- malate-aspartate shuttle primarily functions in the liver, kidney and heart
- KINETICS
72
Complete oxidation of glucose
- yields 30 ATP using the glycerol 3-phosphate shuttle
| - yields 32 using the malate-aspartate shuttle
73
Oxidative phorphorylation regulation
- regulated by cellular needs
- O2 consumption inhibited by the avaliability of ADP as substrate
- mass action ratio is high ATP-ADP is almost fully phosphorylated
- mass action ratio only slightly fluctuates even during extreme energy demand
- ATP is formed only as fast as it is used
Biochemistry II
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