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99:163 Medical Biochemistry Bioenergetics:
Oxidative Phosphorylation & ATP Synthesis
Instructor: Dr. Madeline A. Shea Prof. of Biochemistry 335-7885, 4-450 BSB
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Oxidative Phosphorylation Generally follows organization of Stryer Ch. 18
Introduction to Respiration and Review of Membrane/Protein Terminology 18.1 Oxidative Phosphorylation in Eukaryotes Occurs in
Mitochondria having Endosymbiotic Origins 18.2 Oxidative Phosphorylation is
Dependent on Electron Transfer 18.3 The Respiratory Chain Consists of Four Complexes
Including Three Proton Pumps 18.4 A Proton Gradient Powers the Synthesis of ATP 18.5 Many Shuttles Allow Movement Across the Mitochondrial Membranes 18.6 Regulation of Oxidative Phosphorylation is
Governed Primarily by the Need for ATP
Other sources used for figures - Good resources for studying:
Lehninger Ch. 11 Biological Membranes & Transport, Ch. 19 Oxidative Phosphorylation
Lippincott Review
Molecular Biology of the Cell
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Study Questions & Key Concepts
1. What is oxidation? What is reduction? Give an example of each. 2. What are reduction potentials? How is free energy related to transfer of electrons? 3. Describe oxidative phosphorylation. What are the starting materials and final
products? What proteins are involved? What are their cofactors? What is the energetic driving force of oxidative phosphorylation?
4. With respect to the structure of a mitochondrion, where are the sites of oxidative phosphorylation and the TCA cycle?
5. What is the evidence for the origin of mitochondria now found in eukaryotic cells? 6. How do proteins participate in the electron transfer process? 7. Are the genes for the polypeptides that make up the proton pumps in the electron
transport chain all in the nucleus? 8. Flow of electrons from NADH leads to pumping of proton from which proteins? 9. Describe the path by which electrons from FADH2 enter the electron transport
chain. Why is less ATP made from FADH2? 10. What is the function of protons required for synthesis of ATP by ATP synthase? 11. What evidence demonstrated that the ATP synthase protein complex was rotating
relative to the plane of the membrane? 12. What are uncouplers? Provide an example of when this might be useful.
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Cellular Respiration
An ATP-generating process in which an inorganic compound (such as molecular oxygen, O2) serves as the ultimate electron (e-) acceptor.
Donor can be organic or inorganic.
Good news - it works efficiently. Accounts for 26 of 30 ATP/glucose* Bad news - it requires
MANY steps. Thats also why it works so well.
*This is NOT a universal constant like ! . The value varies depending on conditions and assumptions.
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Going From Food to ATP: Most of the action is in Mitochondria*
ATP
* 1948, Lehninger & Kennedy
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Connections to Metabolism
Carbon fuels oxidized in citric acid cycle inside mitochondrial matrix to make electrons with high transfer potential.
Electron-motive force converted to proton-motive force via transmembrane proteins
Multiple oxidation-reduction centers: quinones, flavins, iron-sulfer (Fe-S) clusters, hemes & copper (Cu).
Ultimately, connected to phosphate-transfer potential via ATP Synthase , an enzyme driven by flow of protons into mitochondrial matrix.
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Architecture of the Mitochondrion Home of Oxidative Phosphorylation & ATP Synthase
Stryer 5e 18.1 Electron
micrograph of a
mitochondrion.
Stryer 5e 18.3 Diagram of a mitochondrion. Organelle: roughly 2 !m by 0.5 !m (actually dynamic in size & shape) mtDNA* = ~17 kbp, 13 proteins, rRNA, tRNA Cristae = large surface area (shorefront property) packed into a compact unit. Outer membrane - very permeable, because of porin or VDAC (Voltage Dependent
Anion Channel), an open beta-barrel structure (open border). Inner membrane: impermeable to ions, polar molecules. Specific transporters
shuttle metabolites - ATP, pyruvate and citrate. They all need a passport. Membrane Potential: Negative/matrix, Positive/cytosolic
*DNA tracks maternally, may be used for lineage or forensic purposes.
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Mitochondria: Related to Bacteria Closest Known Relative is Rickettsia
Rickettsia free-living bacterium that causes louse-born typhus relative of the presumed ancestor of all mitochondria
106 base pairs 834 protein genes
Mitochondria incapable of independent living but have a genome require some cellular proteins do a great job of making ATP vary in size among eukaryotes
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Overlapping Gene Complements of Mitochondria
Gene names indicate known rRNA & protein coding sequences within the organism represented by the oval.
Reclinomonas americana mitochondrial genome has 62 protein-coding genes and contains all those found in all the sequenced mitochondrial genomes.
Single endo-symbiotic event. Students are not responsible for memorizing this set of gene names!
yeast
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Proton Flux
Entry
Prot
on
Flux
Ex
it Positive
Negative
Coupling of Oxidative Phosphorylation To ATP Synthesis Requires Compartments
Oxidation of fuels and ATP synthesis (phosphorylation of ADP) are coupled by transmembrane proton fluxes.
Concentration gradients store energy.
Stryer 5e 18.2
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Review of Membrane/Protein Terminology Lehninger, Ch. 11
Figures from Lehninger, Nelson & Cox!
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Proteins Inserted in Membranes* Control Concentration Gradients
* May Span Lipids with Helices or Sheets
Osmotic Pressure
Outside = periplasm (sometimes cytoplasm)
Inside = matrix
Figures from Lehninger, Nelson & Cox!
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NADH + 11 H+matrix + 0.5 O2 ! NAD+ + 10H+periplasmic + H2O
Net: NADH + H+ + 0.5 O2 ! NAD+ + H2O
Figure from Lehninger, Nelson & Cox!
Overview of Electron Transport Pathway: Electrons from Metabolites to Oxygen
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Transmembrane Proteins Move Electrons According to Energetics of Electro-Chemical Rxns.
In OxPhos*, the electron transfer potential ("Eo) of NADH or FADH2 is converted into the phosphoryl transfer potential ("Go) of hydrolysis of ATP.
"Go = n F "Eo (= -RTlnK) n = # of electrons transferred F = faraday constant = 23 kcal/mol/volt "Eo = difference in reduction potentials
for reactants and products (how steep is the hill between the top and bottom).
* OxPhos = Oxidative Phosphorylation
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Reduction/Oxidation = Redox Redox Couple: X and X- (e)
Reduction/Oxidation mnemonics: LEO says GER or OIL RIG
X = oxidized form X- = reduced form
X- + H+ = X + 0.5 H2 Half-Reactions
X- = X + e- H+ + e- = 0.5 H2
Reference Reduction potential of H+:H2 = 0
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Lab Measurement of Redox Potential
Apparatus for the measurement of the standard oxidation-reduction potential of a redox couple.
Electrons (e-), but not X or X-, can flow through the agar bridge.
Biochemists use pH 7 (hydrogen at 10-7 M) as standard reaction conditions, in contrast to chemists who use 1 M.
Figure from Stryer5e!
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Standard Reduction (Redox) Potentials Oxidant + e- = Reductant
Numbers are provided to demonstrate known range.
0.67 to +0.82 Volts. Students are not responsible for
memorizing the entire list.
Selected reactions will be highlighted during lectures.
Iron GER +3 + e- = 2+
Table from Stryer5e!
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ReDox Example: NADH & FMN
Figures from Lippincott
Loses e- Gains e-
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Redox in Aqueous Solutions Linked to Proton Uptake and Release
Reduction
Oxidation
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Separation Distance & Medium Affects Electron-Transfer Rate
Electrons move faster through proteins than through vacuum.
Log10 of e- transfer per second
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Figure from Stryer!
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Legend: Distance dependence of electron-transfer rate
The electron transfer rate drops as the electron donor and the electron acceptor move apart.
In a vacuum, the rate decreases by a factor of 10 for every increase of 0.8 .
In proteins, the rate decreases more gradually - a factor of 10 for every increase of 1.7 .
This is approximate because variations in the amino acid composition and structure of the protein can affect the rate.
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From Food to ATP in Mitochondria
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Transmembrane Electron Handoffs Highlights Redox Partners, H+, Coupling
Figure from Lehninger, Nelson & Cox!
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Electron flow leads to proton flux by Complexes I, III, and IV
Electron Carriers in Respiratory Chain: e- move from NADH/FADH2 to O2
I =NADH-Q oxidoreductase, or II = Succinate-Q reductase coenzyme Q or ubiquinone -
links chain to the citric acid cycle. III = Q-cytochrome c oxidoreductase cytochrome c - small soluble protein
shuttles electrons from III to IV. IV = cytochrome c oxidase - catalyzes the
final step - reduction of oxygen.
FADH2
NADH
I
II
III
IV
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Transmembrane Electron Handoffs
FADH2
NADH
I
II
III
IV
I III IV
II
Not shown in this illustration
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Legend for Complexes I, III, IV: Electrons move between respiratory enzyme complexes
The figure shows relative size and shape of each complex.
During the transfer of electrons from NADH to oxygen (red lines), ubiquinone and cytochrome c serve as mobile carriers that ferry electrons from one transmembrane protein complex to the next.
Protons are pumped across the membrane by each of these respiratory enzyme complexes, accompanying the redox reactions.
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Mitochondrial Electron-Transport Chain
Numbers provided to demonstrate relative sizes. Students are not responsible for memorizing mass values.
Oxidant + e- = Reductant
Table from Stryer5e!
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Transmembrane Electron Handoffs Highlights Metals in Proteins
Mathews & van Holde
FADH2
NADH
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Purpose of Electron Transport
To capture energy stored in electron-rich compounds coming out of metabolic pathways.
To establish a proton gradient costs about 5 kcal/mol to push a proton across a membrane having "pH of 1.4.
To drive synthesis of ATP indirectly
To drive transport of metabolites across the mitochondrial membrane.
But why is it a favorable process?
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Standard Reduction Potentials Oxidant + e- = Reductant
Positive Voltage = Favorable "G
Oxygen to Water = +0.82 V
Voltage is like money:
Negative Voltage = Unfavorable "G
for reaction
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Favorable Descent = Unfavorable Climb
Walking Uphill in Steep Hill, Lincoln, England
Sliding Downhill (Strawberry Canyon, Berkeley, CA)
Positive Voltage
Negative Voltage
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NADH to O2: Downhill by 1.14 Volts
Standard Reduction Potentials for Half-Reactions a. 0.5 O2 + 2 H + + 2 e- ! H2O E"0 = +0.82 V b. NAD+ + 2 H+ + 2 e- ! NADH + H+ E"0 = -0.32 V*
"E, Net Difference = 1.14 Volts Subtracting b from a c. 0.5 O2 + NADH + H + ! H2O + NAD+ "G = -(2)(23.06)(+0.82) - (-(2)(23.06)(-0.32)) = -52.6 kcal/mol
vs. -7.5 kcal/mol for hydrolysis of ATP
Relay Race with Electrons Energy of Passing Baton "Go = n F "Eo
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NADH-Q oxidoreductase (Complex I*) NADH oxidized to NAD+
> 30 polypeptides - combo of mitochondrial & cellular High potential e- of NADH enter chain, transferred to FMN
(flavin mononucleotide) to give FMNH2. NADH + Q + 5 H+matrix ! NAD + + QH2 + 4 H+cytosol
reduce Q
NADH Ox. Figure from Stryer 5e
Adapted from Lehninger + H+ 2H+
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Legend for Structure of NADH-Q oxidoreducatase (Complex 1)
The structure has been studied by electron microscopy. The model has 22 resolution.
Protein complex consists of a membrane-spanning part and a long arm that extends into the matrix.
NADH is oxidized in the arm, and the electrons are transferred to reduce Q in the membrane.
After N. Grigorieff, J. Mol. Biol. 277 (1998): 1033-1048.
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Stepwise Oxidation of Flavins FMN + 2e- & 2H+ = FMNH2
The reduction of flavin mononucleotide (FMN) to FMNH2 proceeds with concomitant binding of protons.
FMN can also accept just 1 e-, forming a semiquinone intermediate.
Subsequently, electrons are transferred from FMNH2 to Fe-S-containing subunits in NADH-Q oxidoreductase.
Figure from Stryer 5e
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Iron-Sulfur Clusters Undergo Oxidation-Reduction Reactions
A. 1 Fe ion bound by four Cys residues. B. 2Fe-2S cluster with iron ions bridges by sulfide ions. C. 4Fe-4S cluster (cube). Each of these can undergo oxidation-reduction reactions
Iron cycles between Fe2+ (reduced) or Fe3+(oxidized) Found in Complexes of the Electron Transport Chain
A 1Fe C 4Fe B 2Fe
Figure from Stryer 5e
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Oxidation States of Coenzyme Q
isoprenoid tail of Coenzyme Q10
e- +
H+ Oxidized Quinone
Figure from Stryer 5e
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Coupled Electron-Proton Transfer Reactions
Reduction of a quinone (Q) to QH2 results in the uptake of two H+ from the mitochondrial matrix.
Pair of e- on QH2 transferred to Fe-S center and H+ released on cytosolic side. Bound Q & mobile Q account for 4 H +
This is an area of ongoing investigation! Figure from Stryer 5e
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Succinate dehydrogenase (= succinate-Q reductase) is the only membrane-bound part of citric acid cycle.
Cofactors: FAD, 3 Fe-S centers, Heme b & Q
Total path of ~40 each step # 11
Heme b not part of transport but is involved in scavenging e-
Processes similar in mitochondria & bacteria
Complex II: succinate ! Ubiquinone
Figure from Lehninger, Nelson & Cox!
Matrix
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Donor-Acceptor Relationships
For each reaction, keep in mind that you can check for consistency by following electrons and the protons.
Every redox reaction - LEO & GER - like a relay race: One compound Loses an e- One Gains an e- (is reduced)
Goal of the game: get the e- to oxygen!
Figure from Lehninger, Nelson & Cox!
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Q-cytochrome c oxidoreductase (Complex III, cytochrome bc1).
Cytochrome: any e- transfer protein with a heme group.
Homodimer; monomer = 11 distinct polypeptides
Major prosthetic groups: 3 Hemes (L=low, H=high) 1 2Fe-2S cluster
Mediates e-transfer between quinones in the membrane & soluble cytochrome c in the intermembrane space of the mitochondria.
Rieske center: 2 Cys/2 His coordination - stabilizes reduced form
QH2 + 2 Cyt cox + 2 H+matrix ! Q + 2 Cyt cred + 4 H + cytosol
Figure from Stryer 5e
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Figures from Lehninger, Nelson & Cox!
Complex III Moves Q(ubiquinone) to cytochrome C
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Complex III: the Q cycle
Figure from Lehninger, Nelson & Cox!
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Q Cycle (blue/oxidized, red/reduced)
2 e- of bound QH2: 1 to cytochrome c, 1 to bound Q = the semiquinone Q. Newly formed Q dissociates and is replaced by a second QH2, QH2: gives up 2 electrons: 1 to a second molecule of cytochrome c and the
other to reduce Q to QH2. 2nd electron transfer results in the uptake of two protons from the matrix. Prosthetic groups: oxidized = blue and reduced = red.
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Cytochromes c & c1: Attached Hemes
Heme group - recall Myoglobin and Hemoglobin? Cytochromes have the same iron-containing group: protoporphyrin IX.
Heme is covalently attached to cytochrome c through thioether linkages formed by the bonding between sulfhydryl groups of cysteine residues and vinyl groups on protoporphyrin.
Figure from Stryer 5e
From http://chemistry.umeche.maine.edu/CHY431/Evolve2.html
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Cytochrome c Highly Conserved Carrier
Ball-and-stick representation of the side chains for 21 conserved amino acids and the heme group (sideways view of the plane of the protoporphyrin).
See http://chemistry.umeche.maine.edu/CHY431/Evolve2.html
Cyt C: Estimated to be in use than 1.5 billion years ago Conserved 2 &3 Structure: 26/104 residues are invariant All known cyt c sequences will react with cyt c oxidase
Figure from Stryer 5e
www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/cytochrome-c.html#products
Cyt C also also participates in the cytosolic caspase proteolytic cascade of apoptosis
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NADH + 11 H+matrix + 0.5 O2 ! NAD+ + 10H+periplasmic + H2O
Net: NADH + H+ + 0.5 O2 ! NAD+ + H2O
Figure from Lehninger, Nelson & Cox!
Overview of Electron Transport Pathway: Now for the final handoff to Oxygen
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Complex IV: Cytochrome c Oxidase
Enzyme = 13 polypeptide chains (3 mitochondrial, 10 cellular in origin). 3 Major prosthetic groups = CuA/CuA, heme a1 and heme a3 -CuB. Heme a3CuB = site of the reduction of oxygen to water. CO(bb) = carbonyl group of peptide backbone
Heme a1
Heme a3 CuB
CuA/CuA
Matrix
Periplasm
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Complex IV: Moves e- from cytochrome c to O2
Cytochrome oxidase Transfers e- from cyt. c to
oxygen and pumps H+ across membrane
13 subunits (2-heme, 2- sulfur centers)
One e- transferred at a time; partially reduced oxygen remains tightly bound to complex
Figure from Lehninger, Nelson & Cox!
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Cytochrome c Oxidase Mechanism: Steps 1-4 4 Cyt cred + 4 H+matrix + O2! 4 Cyt cox + 2 H2O
Cytochrome c carries only 1 e-. It reduces* targets by being oxidized . 1. electron from Cytochrome c #1 goes to CuB 2. electron from Cytochrome c #2 goes to Fe in heme a3 (Fe3+ to Fe2+) 3. Both Fe and Cu are reduced. 4. Oxygen (O2) binds to Fe2+ in heme a3. *OIL RIG = Oxidation is Loss, Reduction is Gain, blue/oxidized, red/reduced
Cytochrome c Oxidase
Cytochrome c
a
a3 CuB By the Iron
CuA
4 3 2 1
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Cytochrome c Oxidase Step #5: Peroxide Bridge
Addition of electrons Cu2+ (cupric) > Cu+ (cuprous) Fe3+ (ferric) > Fe2+ (ferrous)
Oxygen bound to heme a3 is reduced to peroxide (O22-) by the presence of CUB.
Addition of e- from cyt c & H+ 1. CuB2+-OH 2. Fe4+=O (ferryl)
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Cytochrome c Oxidase Mechanism: Steps 5-8 4 Cyt cred + 4 H+matrix + O2! 4 Cyt cox + 2 H2O
5. Formation of Peroxide Bridge between CuB and Fe in heme a3 6. Cleavage of O-O bond by addition of H+, oxidation of cytochrome c. 7. Addition of H+: reduction of the ferryl group, oxidation of cytochrome c. Addition of final e- and 2 more matrix protons make 2 H2O.
4 protons are pumped without chemical change. Enzyme is regenerated = all prosthetic groups are oxidized -
ready to receive electrons again!
*OIL RIG = Oxidation is Loss, Reduction is Gain, blue/oxidized, red/reduced
5 6 7 8
Cytochrome c Oxidase
Cytochrome c
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Summary of H+ transport by Cytochrome c Oxidase
4 chemical protons are taken from the matrix side to reduce O2 to two H2O.
4 additional pumped protons are transported out of the matrix and released on the cytosolic side in the course of the reaction.
Start: 4 cyt c red + 4 H+matrix + O2 End: 4 cyt c ox + 2H2O; 4 H+ pumped
"G = -55.34 kcal/mol Pumped protons double the efficiency of
free-energy ("G) storage in the form of a proton gradient for this final step in the electron-transport chain.
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Standard Reduction Potentials Oxidant + e- = Reductant
Reactions in this box all have + Voltage = Favorable "G
cyt c (3+) to (2+) = +0.22 V Oxygen to Water = +0.82 V
An oxidant can extract/accept an electron from a donor. e.g., Fe(+3)
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MBoC View of e- from Cytochrome C
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Danger Lurks in the Reduction of O2
Recall: Oxygen (biradical, 2 unpaired electrons) is bound to heme a3 and reduced to peroxide (O22-) by the presence of CuB.
O2 O2- O2
2-
oxygen superoxide peroxide
Superoxide (1 unpaired e-) and Peroxide can be harmful to many cellular components Fe/Cu ions hold onto peroxide to prevent release.
Enzyme SOD (Superoxide Dismutase) traps reactive oxygen species that escape (making oxygen and hydrogen peroxide).
add e- add e-
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Mitochondria and Oxidative Stress
Figure from Lehninger, Nelson & Cox!
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NADH + 11 H+matrix + 0.5 O2 ! NAD+ + 10H+periplasmic + H2O
Net: NADH + H+ + 0.5 O2 ! NAD+ + H2O
Figure from Lehninger, Nelson & Cox!
Overview of Electron Transport
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ATP yield from Glucose
Stryer Table 18.4 Single page at back of handout (more readable)
Note: estimates for the lower half are in flux - they are based on research in progress.
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How to Couple Reduction of Oxygen with ATP Synthesis?
Direct Coupling - we have seen examples of protein-protein interactions going back to Hemoglobin binding oxygen cooperatively, where the action of a ligand on one subunit changes the likelihood of binding to another.
Indirect coupling - Diffusible substances can also couple processes. Consider examples of feedback inhibition that we saw in pathways such as Tryptophan synthesis (DNA binding) or metabolism (feedback inhibition of enzymes).
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Chemiosmotic Hypothesis
Electron transfer through the respiratory chain leads to proton transfer from the matrix to the cytosolic side of the inner mitochondrial membrane.
The pH gradient and membrane potential constitute a proton-motive force (PMF) that drives ATP synthesis.
Wacky indirect coupling earned Peter Mitchell a Nobel Prize
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Testing the Chemiosmotic Hypothesis Is change in [H+] sufficient to drive ATP synthesis?
ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a light-driven proton pump) and ATP synthase are illuminated.
The orientation of ATP
synthase in this reconstituted membrane is the reverse of that in the mitochondrion.
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Structure of ATP Synthase also called ATPase
Fo = proteins in membrane proton conducting unit
Subunit a - single copy, Subunit c - 10 to 14 copies
F1 - proteins in matrix Catalytic - ATP synthesis/breakdown 3 # subunits, 3 $ subunit 1 each of %, &, ' subunits
Bridge: 2 b subunits Stator - a, 2b, &, 3#, 3$( Rotor - c ring, %, ' stalk
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ATP Synthesis Mechanism
ADP binds in P-loop NTPase domains of # & $( catalysis occurs in $ subunits, dependent on Mg2+ Water takes oxygen from orthophosphate.
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ATP Synthesis Does Not Require PMF*, But ATP Release Does
Isotope-exchange experiments indicated that 18O could be incorporated into orthophosphate during cycling of enzyme. This showed that enzyme-bound ATP is formed from ADP & Pi in the absence of a *proton-motive force (PMF).
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ATP Synthase Non-equivalent Nucleotide-binding Sites
% subunit passes through center of #3$3 hexamer. This breaks symmetry.
Nucleotide-binding sites in 3 $ subunits are no longer equivalent. Loose: Binds ADP + Pi Tight: Binds ADP + Pi
converts to ATP Open: can release ATP T
O L %
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%
ATP Synthase: Another View Non-equivalent Nucleotide-binding Sites
Loose
Tight
Open
Lehninger, Ch. 19
Loose: Binds ADP + Pi Tight: Binds ADP + Pi, converts to ATP Open: can release ATP
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T
O L
Binding-change mechanism for ATP synthase
Rotation of the asymmetric % subunit(spindle in the middle) interconverts the 3 $ subunits.
The T (tight) form, which contains newly synthesized ATP that cannot be released, is converted into the O (Open) form.
The O form can release ATP and then bind ADP and Pi to begin a new cycle.
Hypothesis: Synthesis should run motor in one direction and hydrolysis should run motor in opposite direction.
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ATP Release from $ subunit in Open Form
Unlike the Tight and Loose forms, the Open form of the $ subunit can change conformation sufficiently to release bound nucleotides. Both have been observed in crystals.
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NOTE: Only the Beta Subunits Catalyze Conversion of ADP to ATP
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ATP Synthesis vs. Hydrolysis: Motor Direction Depends on Proton Flux
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ATP-driven Rotation in ATP Synthase Experimental Demonstration Using Artifical Tag
Experimental Design: 1. #3$3 hexamer of ATP synthase is fixed to a surface 2. % subunit is projecting upward and linked to a fluorescently labeled actin
filament. This creates a tag that can be visualized.* 3. Addition of ATP, and subsequent hydrolysis result in counterclockwise
rotation of the % subunit, demonstrating coupling. This motion can be seen under a fluorescence microscope.
NOTE: Actin is not a physiological accoutrement of ATP Synthase!
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ATP Synthase: Rotary Engine
Oxidative phosphorylation shows that proton gradients are an interconvertible currency of free energy ("G) in living cells.
Movies courtesy of Yoshida & Toru Laboratories, via Stryer5e website Note: Actin was attached as a molecular baton - to show movement - it is not a naturally occurring partner in the ATP Synthase complex.
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H+-conducting unit of ATP synthase
Each subunit c has 2 # helices that span the membrane. An aspartic acid residue in the second helix is positioned at the center of the membrane.
The subunit a appears to include 2 half-channels that allow protons to enter and pass partway but not completely through the membrane.
a
c
The exact path of the hydrogen ions through the pump is still a matter of intense study, Goodsell
a
c
1c17
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Proton Flux drives Rotation of c Ring
H+ enters from the intermembrane space into the cytosolic half-channel to neutralize the charge on an aspartate residue in a c subunit. With this charge is neutralized, the c ring can rotate clockwise by one c subunit, moving an aspartic acid residue out of the membrane into the matrix half-channel.
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Proton Flux drives Rotation of c Ring
This proton can move into the matrix (following gradient), resetting the system to its initial state.
Full turn of ring of c subunits = 3 ATP synthesized (10 to 14 protons)/(3 ATP) = ~3 to 4 H+/ATP
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Proton Path through Inner Membrane
Subunit a (half-moon shape) holds static (held by b2).
Each proton (H+) from cytosolic intermembrane space
1. enters the cytosolic half-channel,
2. follows a complete rotation of the c ring, and
3. exits through the half-channel into the matrix. a
c c c
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Common Motifs in Protein Structure/Function: ATP Synthase Related to G Proteins (GTPases)
# and $ subunits of ATP synthase have P-loop NTPase sites. Like G-proteins, exchange of NDP/NTP is accompanied by
conformational change which is stimulated by interactions with other proteins.
Example: Sos (yellow) a guanine-nucleotide exchange factor binds to Ras (purple outline) and opens the NTP site, allowing GDP to dissociate and GTP to bind.
Stryer5e Fig. 15.3
Ras
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What limits efficient ATP Synthesis?
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Small Molecules can Uncouple Steps of Oxidative Phosphorylation
2,4-Dinitrophenol, a lipid-soluble compound, can carry protons (H+) across the inner mitochondrial membrane.
The dissociable proton is shown in red.
Membrane + H+
Membrane
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Proteins can Uncouple e- & H+
Uncoupling protein-1 (UCP1) generates heat by permitting the influx of protons into the mitochondria without the synthesis of ATP.
Means of generating heat in newborn animals/infants. Brown adipose tissue has mitochondria rich in UCP-1.
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Chemical Inhibitors of Electron Transport Uncouple Separable Steps in Pathway
I - Rotenone and Amytol selectively inhibit NADH-Q oxidoreductase, (but do not affect entry of e- from II, succinate-Q reductase).
III - Antimycin A interferes with e- flow from cyt bH in Q-cytochrome c reductase.
IV - Cyanide and azide bind to ferric (3+) form of heme in cytochrome c oxidase, while carbon monoxide binds to the ferrous form (2+).
Useful biochemically (in vitro), but deadly physiologically
I
III
IV
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Next we consider how mitochondria transport ions (ADP/ATP, metabolites) across membranes
Figures from Lehninger, Nelson & Cox!
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99:163 Medical Biochemistry Mitochondrial Transport & Shuttles
Instructor: Dr. Madeline A. Shea Prof. of Biochemistry 4-450 BSB, 335-7885
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Study Questions & Key Concepts
1. How are mitochondria distributed in cells? What shape(s) do they adopt? Where are ox-phos electron carriers located?
2. Why do mitochondria need transporters and shuttles to move metabolites between the cytosol and the mitochondrial matrix?
3. What pathways or cycles give rise to cytosolic NADH and mitochondrial matrix NADH?
4. Explain the mitochondrial glycerol 3-phosphate shuttle. 5. Explain how the malate-aspartate shuttle works and why it is
needed. How do electrons from the cytosol enter mitochondria? 6. Explain how the ATP-ADP translocase works. What drives the
direction of flow of ATP, and ADP?!7. What mitochondrial protein is a signal for apoptosis to begin?!8. How does the mitochondrial genome differ from the nuclear
genome?!
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Mitochondrial Transport Stryer Ch. 18, Alberts Ch. 14, Lippincott Ch. 5
Electron micrograph of surface of mitochondrial inner membrane Proteins crammed together
100 ATP per second per ATPase
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Legend for MBOC Figure showing energy-generating metabolism in mitochondria
Pyruvate and fatty acids enter the mitochondrion (bottom) and are broken down to acetyl CoA. The acetyl CoA is then metabolized by the citric acid cycle, which reduces NAD+ to NADH (and FAD to FADH2, not shown).
In the process of oxidative phosphorylation, high-energy electrons from NADH (and FADH2) are then passed along the electron-transport chain in the inner membrane to oxygen (O2). This electron transport generates a proton gradient across the inner membrane, which is used to drive the production of ATP by ATP synthase.
The NADH generated by glycolysis in the cytosol also passes electrons to the respiratory chain (not shown). Since NADH cannot pass across the inner mitochondrial membrane, the electron transfer from cytosolic NADH must be accomplished indirectly by means of one of several shuttle systems that transport another reduced compound into the mitochondrion.
After being oxidized, this compound is returned to the cytosol, where it is reduced by NADH again.
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Remember the Chemiosmotic Hypothesis?
All well and good -- as long as other molecules can get into and out of the matrix without disrupting the pH gradient that drives ATP synthesis.
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Views of Mitochondria
Outer membrane very permeable because of porin - also called VDAC (Voltage Dependent Anion Channel).
Inner membrane impermeable to ions, polar molecules. Specific transporters shuttle metabolites.
7
Legend for Mitochondrial Properties
8
Mitochondria are Flexible & Dynamic
Mitochondria change shape often (not stiff grapes). Videomicroscopy of yeast at 3 min. intervals
showed constant fission/fusion reactions.
9
Mitochondria are Often Associated with Microtubules
Stained mitochondria (in top, A)
align along microtubules (bottom, B)
providing energy for cell motility and being moved in cell.
10
Mitochondria are Good Neighbors
Cardiac muscle & sperm tail are positions of high ATP consumption. Mitochondria (ATP engines) are brought to the sperm tail by microtubules. They fuse to make larger coils that wrap the axoneme.
How does fuel get to the engine?
11
Transporters and Shuttles
Electrons from NADH - Glycerol 3-Phosphate Shuttle (general) Malate-Aspartate Shuttle (heart & liver)
Dicarboxylate Carrier (Malate/Phosphate) Tricarboxylate Carrier (Citrate,H+/Malate) Pyruvate (OH-) Phosphate (OH-)
ATP-ADP Translocase
12
NADH: Mitochondrial & Cytosolic
Citric Acid cycle creates NADH within matrix. Glycolytic Pathway creates cytosolic NADH. How does NADH get into mitochondria when inner
membrane is impermeable? How does cell regenerate NAD+ it needs to continue glycolysis?
13
Re-oxidize NADH by Taking its e-
Example: Glycerol 3-Phosphate Shuttle Electrons from cytosolic NADH enter the mitochondria by being used to reduce dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate via cytosolic glycerol 3-phosphate dehydrogenase .
At Cytosol/Matrix Boundary Resulting glycerol 3-phosphate is reoxidized by
electron transfer to an FAD prosthetic group in a membrane-bound mitochondrial glycerol 3-phosphate dehydrogenase.
Subsequent electron transfer from FADH2 to membrane-localized coenzyme Q (ubiquinone) to form QH2 allows these electrons to enter the electron-transport chain.
FADH2
NADH
14
NADH: Glycerol 3-Phosphate Shuttle Smuggling Electrons into Mitochondria
Glycolytic Intermediate
15
Legend for Glycerol 3-phosphate shuttle
Electrons from NADH can enter the mitochondrial electron transport chain by being used to reduce dihydroxyacetone phosphate to glycerol 3-phosphate.
Glycerol 3-phosphate is reoxidized by electron transfer to an FAD prosthetic group in a membrane-bound glycerol 3-phosphate dehydrogenase.
Subsequent electron transfer to Q to form QH2 allows these electrons to enter the electron-transport chain after complexes I and II.
Note - proton pumping of complex I does not occur.
16
But, theres no free lunch (there is always a cost to exchange currency)
FAD as the electron acceptor in mitochondrial glycerol 3-phosphate dehyrogenase allows e- from NADH to be transported against an NADH gradient.
Glycerol 3-phosphate shuttle allows muscle to sustain a high rate of oxidative phosphorylation. However - the price is one ATP per 2 e-, because the process skips proton pumping by NADH-Q oxidoreductase.
Bottom line: Still a good deal because no pumping of NADH/NAD+ is necessary.
17
ATP Yield From Oxidation of Glucose
Numbers provided to demonstrate calculation of yield.
30 ATP per glucose. Recognize that these numbers
include some assumptions about the cost of restoring or maintaining gradients.
Note that there is a difference between starting with FADH2 rather than NADH, resulting in lower efficiency when FADH2 is used.
18
NADH e- in Heart and Liver Move via the Malate-Aspartate shuttle
Electrons from cytosolic NADH enter mitochondria via the malate-aspartate shuttle.
Shuttle = 2 membrane carriers + 4 enzymes. Readily reversible:
The shuttle runs if the NADH/NAD+ ratio in the cytosol is higher than it is in the mitochondria (indicating a build up of NADH electron donors).
This pathway can be seen as moving the bus (malate/aspartate) and/or the passengers (e-)
19
NADH: Malate-Aspartate Shuttle
1. Oxaloacetate + NADH + H+ = malate + NAD + 2. Malate crosses membrane via carrier protein. 3. Reverse reaction occurs. But, oxaloacetate is stuck (not permeable). 4. Oxaloacetate + Glutamate =
Aspartate (which has a passport) + !-Ketoglutarate.
not permeable
Malate Dehydrogenase (from citric acid cycle)
Aspartate Aminotransferase transfers !-amino group to !-ketoacid
deamination
transamination
20
How do mitochondria transport ions (metabolites, ADP/ATP) rather than e- across inner membrane?
Figures from Lehninger, Nelson & Cox!
21
Transporters and Shuttles
Electrons from NADH - Glycerol 3-Phosphate Shuttle (general) Malate-Aspartate Shuttle (heart & liver)
Dicarboxylate Carrier (Malate/Phosphate) Tricarboxylate Carrier (Citrate,H+/Malate) Pyruvate (OH-) Phosphate (OH-)
ATP-ADP Translocase
22
Antiporters & Symporters
Antiporters transport 2 compounds in opposite
directions make use of downhill flow of one
compound to uphill flow of another. Like a pulley - falling weight lifts
another.
Symporters Transport 2 compounds together. Uses the flow of one compound to
bring another along.
23
Mitochondrial Transporters
Transporters (carriers) are transmembrane proteins that move ions and charged metabolites across the inner mitochondrial membrane.
Dicarboxylate: malate, succinate and fumarate can be transported in exchange for phosphate.
Tricarboxylate: Citrate and H+ are exchanged for malate.
24
Transport of Charged Species Linked to gradients of voltage and pH
25
Transporters and Shuttles
Electrons from NADH - Glycerol 3-Phosphate Shuttle (general) Malate-Aspartate Shuttle (heart & liver)
Dicarboxylate Carrier (Malate/Phosphate) Tricarboxylate Carrier (Citrate,H+/Malate) Pyruvate (OH-) Phosphate (OH-) ATP-ADP Translocase
26
Respiratory Control Dont commit O2 unless ATP Needed
Electrons are transferred to O2 only if ADP is concomitantly phosphorylated to ATP.
27
ADP & ATP are Highly Charged Energy Barrier for Membrane Translocation
Provides opportunity for control & coupling. ATP-ADP Translocase facilitates crossing.
dimer of identical subunits, ~ 30kDa abundant protein, accounts for one-sixth of protein in the
inner mitochondrial membrane single site for nucleotide (at dimer interface). similar affinity for ATP and ADP ATP released from translocase (to cytosol) faster than ADP is
released (to matrix) because it has more negative charge. Crossing is costly
Charge of 2- of ADP vs. 3- of ATP means ATP release breaks down the positive potential. Approx. 1/4 of energy yield from e- transfer by respiratory chain is consumed to regenerate the membrane potential lost by exchange of ATP for ADP.
Translocase works like an hourglass - one-for-one exchange.
28
Mitochondrial ATP-ADP Translocase rocking banana model of transport
Coupled entry of ADP into matrix and exit of ATP from the matrix is driven by membrane potential. The conformational change corresponding to eversion of the nucleotide binding site is not known.
2c3e.pdb
29
Transporters are Ancient Molecules Found in Bacteria & Archaea, too.
Lactose Permease: uses proton gradient to drive uptake of lactose (sugar) into E. coli.
30
Secondary Structure & Organization of Mitochondrial Transporters
Many mitochondrial transporters consist of three similar 100-residue units. These units contain 2 putative membrane-spanning segments, for total of 6.
31
1st Transporter Structure was Lac Permease 3.5 1pv6.pdb
Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., Iwata, S.: Structure and Mechanism of the Lactose Permease of Escherichia Coli Science 301 pp. 610 (2003)
32
Proteins Need to Traffic, too. Expulsion of mitochondrial Proteins = Crisis
Cytosolic proteins enter, but mitochondiral proteins do not leave except under conditions of apoptosis (programmed cell death). Cytochrome c is a potent activator of apoptosis. When in the cytosol, it triggers the activity of proteases called caspases. These cysteine proteases are highly conserved across species and have specific cellular targets such as structural proteins or DNA management enzymes.
33
Genes in Mitochondrial DNA
mt DNA code is different from standard eukaryotic code. UGA is a universal STOP codon that is used for Trp in mitochondrial genes (see Table 14-3 in Alberts et al, MBOC).
Rate of mutation is ~10-fold higher than mammalian DNA. Diseases related to mutations in mtDNA are being recognized -
Leber hereditary optic neuropathy (LHON), a form of blindness occurs in middle age because of mutations in NADH-Q oxidoreductase.
mt Ribosomes are larger than prokaryotic ribosomes, and have higher protein:RNA ratio, and less RNA
2 rRNA 22 tRNA 13 proteins
34
Mitochondria: Essential Subcontractor, Master of Thermodynamics & Chemical Work
Copyright 2005 Lippincott Williams & Wilkins
Figure 6.17
Reducedsubstrate
O2
ADP + Pi ATPH2O
Oxidizedsubstrate
Summary of key concepts for oxidative phosphorylation. [Note: Electron flow and ATPsynthesis are are envisioned as sets of interlocking gears to emphase the idea of coupling.]
Oxidative phosphorylation
comprised of
TCA cycle and -Oxidation of
fatty acids
An electrical and a pH gradient
The inner mitochondrial membrane
Protons
Reenter the mitochondrial matrix
Passing through a channel in the ATP synthetase molecule
Notablebecause
Inner mitochondrial
membrane
across
to
by
resulting in
allowing
NADH and FADH2
Electron transport chain
coupled with
Electron flow
donate electrons to
produce
leads to
from
FMNCoQCytochrome bCytochrome cCytochrome a + a3
visualized as
visualized as
Only component that can react directly with oxygen
Rich in proteinImpermeable to most small moleculesContains transporters for specific compounds
The synthesis of ATP from ADP + Pi
e
e e e e e
Transport of protons (H+)
creating
MITOCHONDRIALMATRIX
The matrix to theintermembrane space
STRYER 5e
TABLE 18.4 ATP yield from the complete oxidation of glucose
Reaction sequenceATP yieldper glucosemolecule
Glycolysis: Conversion of glucose into pyruvate(in the cytosol)
Phosphorylation of glucose -1Phosphorylation of fructose 6-phosphate -1Dephosphorylation of 2 molecules of 1,3-BPG +2Dephosphorylation of 2 molecules of phosphoenolpyruvate +22 molecules of NADH are formed in the oxidation of 2 molecules of glyceraldehydes 3-phosphate
Conversion of pyruvate into acetyl CoA(inside mitochondria)
2 molecules of NADH are formedCitric acid cycle (inside mitochondria)
2 molecules of guanosine triphosphate are formed from 2 molecules of succinyl CoA +2
6 molecules of NADH are formed in the oxidation of 2 molecules each of isocitrate, -ketoglutarate, and malate2 molecules of FADH2 are formed in the oxidation of 2 molecules of succinate
TABLE 18.4 ATP yield from the complete oxidation of glucose
Reaction sequenceATP yieldper glucosemolecule
Oxidative phosphorylation (inside mitochondria)2 molecules of NADH formed in glycolysis; each yields 1.5
molecules of ATP (assuming transport of NADH by theglycerol 3-phosphate shuttle) +3
2 molecules of NADH formed in the oxidative decarboxylation of pyruvate; each yields 2.5 molecules of ATP +52 molecules of FADH2 formed in the citric acid cycle; each yields 1.5 molecules of ATP +36 molecules of NADH formed in the citric acid cycle; each yields 2.5 molecules of ATP +15
NET YIELD PER MOLECULE OF GLUCOSE +30
Source: The ATP yield of oxidative phosphorylation is based on values given in P.C. Hinkle, M.A.Kumar, A. Resetar, and D.L. Harris, Biochemistry 30 (1991): 3576.Note: The current value of 30 molecules of ATP per molecule of glucose supersedes the earlier oneof 36 molecules of ATP. The stoichiometries of proton pumping, ATP synthesis, and metabolitetransport should be regarded as estimates. About two more molecules of ATP are formed permolecule of glucose oxidized when the malate-asparate shuttle rather than the glycerol 3-phosphateshuttle us used.
A mitochondrionImage: Wikipedia
The Scientist: NewsBlog:First fix for mitochondrial diseasesPosted by Victoria Stern[Entry posted at 26th August 2009 06:00 PM GMT]View comments(4) | Comment on this news story
Researchers have for the first time succeeded in replacing defective mitochondrial genomes with healthy ones in monkeyembryos--a technique that could be used to prevent children from inheriting a variety of incurable genetic diseasescaused by defective mitochondrial genes, they report online today (August 26) in Nature.
"The general idea of preventing mitochondrial diseases by altering egg cells has been aroundfor quite a while now," David Samuels, a professor of molecular physiology and biophysics atVanderbilt University School of Medicine, told The Scientist in an email. "The difficulty hasbeen in working out how to actually carry out the procedure without harming the egg cell,"added Samuels, who was not involved in the study.
Mitochondria, which generate most of the cell's energy supply, contain their own genome,distinct from the cell's nuclear DNA, which is inherited exclusively through the mother.Mutations in mitochondrial DNA can deplete cells of energy and eventually kill them.Mitochondrial genome defects are associated with numerous diseases, including types ofdiabetes and deafness, a form of blindness called Leber's hereditary optic neuropathy, andmetabolic disorders that cause liver failure.
Shoukhrat Mitalipov and his colleagues from Oregon National Primate Research Center devised a way to replace thatdefective DNA by combining in vitro fertilization with cell surgery to generate functional eggs in rhesus monkeys. First,they removed the nucleus from a donor egg cell and replaced it with the nucleus--including nuclear DNA--from themother's egg cell. They then fertilized the egg with the father's sperm, creating an oocyte which contains the parents'nuclear genes and another female's healthy mitochondrial genes.
"Mitalipov's group [was] able to find a time in the egg cell's development when the nuclear DNA and mitochondrial DNAare safely separated, so that they could pull the nuclear DNA out of the egg cell without also pulling out any detectibleamount of the mitochondrial DNA," Samuels said.
After transplanting 15 manipulated embryos into nine rhesus monkeys, the scientists found that the reconstructed eggsfunctioned normally, supporting healthy fertilization and embryo development. Three of the nine rhesus macaquesbecame pregnant, the first giving birth to twins by caesarean section on April 24 of this year.
"So far, we have produced four infants from this method and they are all healthy," Masahito Tachibana, an author on thestudy, said in a telephone press briefing. He said that the group hopes to take the approach to clinical trials in a fewyears.
"It is important to stop transmission of these [mitochondrial] mutations," Tachibana said.
Samuels pointed out, however, that the technique does nothing to help those who already have inherited pathogenicmitochondrial DNA or who already have a mitochondrial disease.
Additionally, researchers do not fully understand the implications of transferring one person's mitochondrial genes into adifferent nuclear background. "This study was very well done, and the data look very convincing," said M. Flint Beal, aprofessor of neurology and neuroscience at the Weill Medical College of Cornell University, who was not involved in theresearch. He added that it provides the first real possibility of preventing mitochondrial diseases. But, he cautioned,"[t]here may be unexpected interactions between the nuclear DNA and mitochondrial DNA."
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[20th September 2007]Support for mtDNA aging theory
[10th April 2006]UK grants mitochondrial license
[16 September 2005]
1 of 3 http://www.the-scientist.com/blog/print/55925/
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