Oxidative Phosphorylation

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  • Created by: LBCW0502
  • Created on: 23-12-17 13:05
The energy of oxidation drives what process?
The synthesis of ATP
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Where does oxidative phosphorylation occur in eukaryotes?
Mitochondria (membrane)
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Oxidative phosphorylation involves the reduction of what?
Oxygen to water (with electrons donated to NADH and FADH2)
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Outline the chemiosmotic theory
Transmembrane differences in proton concentration are the reservoir for the energy extracted from biological oxidation
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Give three features of the oxidative phosphorylation mechanism
Involves flow of electrons, exergonic reaction coupled with endergonic reaction (conservation) and flow of protons through channels provides free energy for synthesis of ATP
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Where are the large functional complexes (for oxidative phosphorylation) located?
In the inner mitochondrial membrane
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Describe the outer membrane of the mitochondria
Freely permeable to small molecules and ions (Mr < 5000)
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Describe the inner membrane of the mitochondria (5)
Impermeable to most small molecules and ions including hydrogen ions. Contains respiratory electron carriers (complexes I-IV), ATP-ATP translocase, ATP synthase (F0F1) and other membrane transporters
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What substances are in the mitochondrial matrix (8)?
PDH, TCA cycle enzymes, fatty acid beta-oxidation enzymes, amino acid oxidation enzymes, DNA/ribosomes, many other enzymes, ATP/ADP/Pi/Mg/Ca/K, many soluble metabolic intermediates
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What do mitochondria resemble to?
Gram negative bacteria (due to having two membranes)
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What is the first step in oxidative phosphorylation?
Dehydrogenases collect electrons from catabolic pathways. Electrons enter the respiratory chain. Electrons are funnelled into universal electron acceptors (NAD/NADP or FMN/FAD)
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Which type of enzymes catalyse reversible reactions involving the conversion of a reduced substrate to an oxidised substrate?
Nicotinamide nucleotide-linked dehydrogenases
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NAD linked hydrogenases remove how many Hs from atoms in the substrate?
2 (one is transferred as a hydride ion to NAD+ and the other is released as a proton in the medium
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Are NADH and NADPH water soluble electron carriers that associate reversibly with dehydrogenases?
Yes
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Which electron carrier supplies electrons to catabolic reactions?
NADH
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Which electron carrier supplies electrons to anabolic reactions?
NADPH
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What do flavoproteins contain?
A very tightly sometimes covalently bound flavin nucleotide (FMN or FAD)
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Oxidised flavin nucleotide can accept how many electrons?
One (semiquinone form) or two (FADH2 or FMNH2)
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Why does electron transfer occur with flavoproteins?
The flavoprotein has a higher reduction potential than the compound oxidised
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What does the standard reduction potential of a flavin nucleotide depend on?
The protein which it is associated
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What is the consequence of local interactions with functional groups in the protein
Leads to the distortion of electron orbitals in the flavin ring, changing the relative stabilities of oxidised and reduced forms
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Where is the flavin nucleotide located in a flavoprotein?
In the active site
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Can flavoproteins serve as intermediates?
Yes
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Which type of reactions involve two electrons being donated?
Dehydrogenation reactions
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Which reactions involve only one electron being accepted?
Reduction of a quinone to a hydroquinone
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Are the complexes in the mitochondrial respiratory chain intrinsic or extrinsic?
Intrinsic (integral) proteins - contain prosthetic groups
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What are the three types of electron transfers in oxidative phosphorylation?
Direct transfer of electrons (reduction of iron from 3+ to 2+), transfer of hydrogen atom (proton + electron) and transfer as a hydride ion
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What is the term 'reducing equivalent' used for?
To designate a single electron equivalent transferred in an oxidation-reduction reaction
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What are the three other types of electron carrying molecules which function in the respiratory chain?
A hydrophobic quinone (ubiquinone) and two different types of iron containing proteins (cytochromes and iron sulfur proteins)
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What is Ubiquinone?
Also called coenzyme Q. Lipid soluble benzoquinone with a long isoprenoid side chain. Able to diffuse (small). Two electron donor/one electron acceptor
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What are cytochromes?
Proteins with characteristic strong absorption of visible light due to their iron containing heme prosthetic groups
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What are the three classes of cytochromes?
a, b and c (have differences in light absorption spectra). Each have reduced iron and three absorption bands in visible range (600, 560, 550)
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Are the heme factors for cytochromes a and b covalently bonded to associated proteins?
No
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Are the hemes in cytochrome c covalently attached through cystine residues?
Yes
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Are cytochromes a, b and some of c, integral proteins of internal mitochondrial membrane?
Yes
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What is the exception for cytochrome c in mitochondria?
A soluble protein that associates through electrostatic interactions with the outer surface of the inner membrane
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Is iron present in the heme of iron-sulfur proteins?
No. Iron is in association with inorganic sulfur atoms or sulfur atoms from cystine residues in the protein
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Describe the structure of Heme A
Porphyrin (four five-membered, nitrogen-containing rings in a cyclic structure). Long isoprenoid tail attached. Conjugated system (accounts for absorption of visible light by heme). Fe ion centre
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Describe the structure of iron protoporphyrin IX (in cytochrome B)
Porphyrin (four five-membered, nitrogen-containing rings in a cyclic structure). Fe ion centre. Found in Hb and myoglobin. Conjugation
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Describe the structure of Heme C
Porphyrin (four five-membered, nitrogen-containing rings in a cyclic structure). Fe ion centre. Heme c is covalently bound to the protein of cytochrome c through thioether bonds to two Cys residues. Conjugation
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What are Rieske iron-sulfur proteins?
One Fe atom is coordinated to two His residues rather than two Cys residues
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Do all iron-sulfur proteins participate in one electron transfers?
Yes (one iron atom of the iron-sulfur cluster is oxidized or reduced)
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How many iron-sulfur proteins function in the mitochondrial electron transfer?
8
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What is the variation in the reduction potential for iron-sulfur proteins?
-0.65 V to +0.45 V (depends on microenvironment of iron within protein)
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Outline the movement of electrons in the respiratory chain
Electrons move from NADH, succinate or another primary electron donor through flavoproteins, ubiquinone, iron-sulfur proteins, cytochromes and then to oxygen
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How is the standard reduction potential of individual electron carriers determined?
Experimentally
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What would be the prediction for the standard reduction potential across the electron carriers?
Carriers function in order of increasing reduction potential (electrons flow from carriers with a low standard reduction potential to carriers with a high standard reduction potential)
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What is the order of the carriers which was determined by the prediction for the flow of electrons?
NADH, Q, cytochrome b, cytochrome c1, cytochrome c, cytochrome a, cytochrome a3, oxygen
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Is the order of standard reduction potentials the same as the actual standard reduction potentials under cellular conditions?
No. Depends on the concentration of reduced and oxidised forms
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Describe a second method for determining the sequence of electron carriers
Reduce the entire chain of carriers by providing an electron source but no electron acceptor (oxygen). The rate at which the electron carriers are oxidised reveals the order in which the carriers function
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What can be concluded from the second method for determining the sequence of electron carriers?
The carrier nearest to oxygen (at the end of the chain) gives up electrons first, followed by the second carrier chain from the end being oxidised etc.
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What is the third method used to determine the sequence of electron carriers?
Use of inhibitors to prevent the flow of electrons through the chain in order to measure the extent of oxidation for each carrier
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What can be concluded from the third method for determining the sequence of electron carriers?
In the presence of oxygen, carriers functioning before inhibited step are reduced and carriers functioning after step are oxidised. Same conclusions from other 2 methods obtained when using several inhibitors to block separate steps
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What can be used to show the resolution of the four electron carrier complexes in the inner mitochondrial membrane?
Gentle treatment of detergent
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What do complexes I (NADH) and II (succinate) catalyse?
The electron transfer to ubiquinone from two different electron donors
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Complex III carries electrons from reduced ubiquinone to where?
Cytochrome c
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How does complex IV complete the sequence?
By transferring electrons from cytochrome c to oxygen
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Describe features of complex I
Called NADH:ubiquinone oxidoreductase or NADH dehydrogenase. Large enzyme composed for 42 different polypeptide chains, contains FMN flavoprotein and 6 iron-sulfur centres. L-shaped (membrane/matrix)
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Complex I catalyses which simultaneous reactions and coupled reactions?
Exergonic transfer of ubiquinone of hydride ion from NADH and a proton from the matrix and the endergonic transfer of the four protons from the matrix to the intermembrane space. Complex I is a proton pump driven by energy of electron transfer
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Why does complex I catalyse a vectorial reaction?
It moves protons in a specific direction from one location (the matrix, which becomes negatively charged with the departure of protons) to another (the intermembrane space, which becomes positively charged)
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Give examples of inhibitors of Fe-S centres of complex I to ubiquinone
Amytal (barbiturate), rotenone (used as insecticide), piericidin A (antibiotic) - block process of oxidative phosphorylation
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What is ubiquinol (QH2) oxidised to?
Oxidised to Q in process involving outward movement of H+
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Describe features of complex II
Succinate to ubiquinone. Succinate dehydrogenase (membrane-bound in TCA cycle), contains 5 prosthetic groups of 2 types and 4 different protein subunits
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Are subunits C and D integral proteins?
Yes - each with three transmembrane helices
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What do the subunits C and D contain?
Heme b and a binding site for ubiquinone (final electron acceptor in the reaction catalyzed by complex II)
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Describe features of subunits A and B
Extend into matrix (or cytosol of bacteria). Contain 3 2Fe-2S centres, bound FAD and a binding site for succinate
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What is a reasonable distance for rapid electron transfer?
11 Angstrom (path of electron from succinate binding site, FAD, Fe-S centres, to Q binding site is 40 Angstrom)
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What is the function of heme b in complex II?
Not in the direct path of electron transfer. Reduces the frequency with which electrons “leak” out of the system, moving from succinate to molecular oxygen to produce the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and the superoxide, O2-
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Other substrates for mitochondrial dehydrogenases pass electrons into the respiratory chain at which level?
At the level of ubiquinone, but not through complex II
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Describe the beta oxidation of fatty acyl CoA
Catalysed by flavoprotein acyl-CoA dehydrogenase. Transfer of electrons from substrate to FAD of dehydrogenase to electron transferring flavoprotein to ETF:ubiquinone oxidoreductase to respiratory chain (reduce ubiquinone)
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Glycerol-3-phosphate is formed from which two methods?
Glycerol released by triacylglycerol breakdown or by the reduction of dihydroxyacetone phosphate from glycolysis (oxidized by glycerol 3-phosphate dehydrogenase)
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Describe features of glycerol-3-dehydrogenase
Flavoprotein on outer surface of inner mitochondrial membrane, channels electrons into the respiratory chain by reducing ubiquinone
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What is the important role of glycerol-3-dehydrogenase?
Shuttling reducing equivalents from cytosolic NADH into the mitochondrial matrix. The effect of electron-transferring enzymes is to contribute to the pool of reduced ubiquinone. QH2 from all these reactions is reoxidized by Complex III
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Describe features of complex III
Cytochrome bc1 complex/ubiquinone:cytochrome c oxidoreductase. Couples the transfer of electrons from ubiquinol (QH2) to cytochrome c with the vectorial transport of protons from the matrix to the intermembrane space
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How were the structures of complexes III and IV determined?
By using x-ray crystallography
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What is the Q cycle?
Accommodates the switch between the electron carrier ubiquinone and the one electron carriers (cytochromes b562, b566, c1, and c). Explains the measured stoichiometry of four protons translocated per pair of electrons passing through the complex III
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What is the net transfer in the Q cycle?
QH2 is oxidized to Q and two molecules of cytochrome c are reduced
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What happens when the single heme from cytochrome c accepts an electron from complex III?
Cytochrome c moves to Complex IV to donate the electron to a binuclear copper centre
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Describe features of complex IV
Cytochrome oxidase. Carries electrons from cytochrome c to molecular oxygen, reducing it to water. Large enzyme, 13 subunits
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What does mitochondrial subunit II contain?
Two copper ions (CuA) complexed with SH groups of two Cys residues in a binuclear centre (resembles 2Fe-2S proteins)
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What does mitochondrial subunit I contain?
2 heme groups, designated a and a3, and another copper ion (CuB). Heme a3 and CuB form a second binuclear centre that accepts electrons from heme a and transfers them to O2 bound to heme a3. Complex IV, cytochrome c, CuA, heme a, heme a3–CuB, O2
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For every four electrons passing through the complex, the enzyme consumes how many substrate H+ from the matrix (N side) when converting O2 to 2H2O?
4 (also uses energy to pump a proton outwards into intermembrane space (P side) for each electron that passes through, adding to the electrochemical potential produced by redox-driven proton transport through complexes I and III
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Is the transfer of two electrons to NAD endergonic or exergonic?
Exergonic
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The standard free energy change is based on what assumption?
Equal concentrations (1 M) of NADH and NAD+
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For each pair of electrons transferred to oxygen, how many protons are pumped out the complexes?
4 protons pumped out of complex I, 4 by complex III and two by complex IV
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What is the proton motive force?
Energy stored (gradient). Chemical potential energy (difference in conc. of chemical species (H ) in 2 regions separated by membrane). Electrical potential energy (results from separation of charge when H+ moves across membrane without counterion
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What is the free energy change for pumping protons outwards?
20 kJ/mol (also - transfer of two electrons from NADH to O2 is accompanied by outward pumping of 10 H+, so 200 kJ of the 220 kJ released by oxidation of a mole of NADH is conserved in the proton gradient
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Do protons flow spontaneously down the electrochemical gradient?
Yes - energy is made available to do work
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Describe the alternative mechanism plant mitochondria has for oxidising NADH
Transfer electrons from ubiquinone to oxygen (by-pass complexes). Cyanide-resistant NADH oxidation
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Card 2

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Where does oxidative phosphorylation occur in eukaryotes?

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Mitochondria (membrane)

Card 3

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Oxidative phosphorylation involves the reduction of what?

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Card 4

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Outline the chemiosmotic theory

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Card 5

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Give three features of the oxidative phosphorylation mechanism

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