overview of oxidative phosphorylation. a. complexes i-iv electrons flow through the etc components...
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Overview of Overview of oxidative phosphorylationoxidative phosphorylation
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A. Complexes I-IVA. Complexes I-IV
• Electrons flow through the ETC components in the direction of increasing reduction potentials
NADH (strong reducing agent, Eo’ = -0.32 volts)O2 (terminal oxidizing agent, Eo’ = +0.82 volts)
• Mobile coenzymes: ubiquinone (Q) and cytochrome c serve as links between ETC complexes
• Complex IV reduces O2 to water
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Complex IComplex I
• NADH-ubiquinone oxidoreductase (NADH dehydrogenase)
• Transfers electrons from NADH to Q
• NADH transfers a two electrons as a hydride ion
(H:-) to FMN
• Electrons are passed through Complex I to Q via FMN and iron-sulfur proteins
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Electron transfer and proton Electron transfer and proton flow in Complex Iflow in Complex I
• Reduction of Q to QH2 requires 2 e-
• About 4 H+ translocated per 2 e- transferred
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Iron in metalloenzymesIron in metalloenzymes
• Iron undergoes reversible oxidation and reduction:
Fe3+ + e- (reduced substrate)
Fe2+ + (oxidized substrate)
• Enzyme heme groups and cytochromes contain iron
• Nonheme iron exists in iron-sulfur clusters (iron is bound by sulfide ions and S- groups from cysteines)
• Iron-sulfur clusters can accept only one e- in a reaction
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Iron-sulfur clustersIron-sulfur clusters
• Iron atoms are complexed with an equal number of sulfide ions (S2-) and with thiolate groups of Cys side chains
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FAD and FMNFAD and FMN(Riboflavin and its coenzymes)(Riboflavin and its coenzymes)
N
N
N
N
O
O
H
CH2
CHOH
CHOH
CHOH
CH2OH
isoalloxazine
ribitol
N
N
N
N
O
O
H
CH2
CHOH
CHOH
CHOH
CH2
O
P
O
O O P
O
O
ON
N
N
N
NH2
HO OH
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Reduction, reoxidation of FMN or FADReduction, reoxidation of FMN or FAD
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Complex IIComplex II
• Succinate-ubiquinone oxidoreductase (or succinate dehydrogenase complex)
• Accepts electrons from succinate and catalyzes the reduction of Q to QH2
• FAD of II is reduced in a 2-electron transfer of a hydride ion from succinate
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Electron transfer in Complex IIElectron transfer in Complex II
• Complex II does not contribute to proton gradient, but supplies electrons from succinate
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Complex IIIComplex III
• Ubiquinol-cytochrome c oxidoreductase
• Transfers electrons to cytochrome c
• Oxidation of one QH2 is accompanied by the translocation of 4 H+ across the inner mitochondrial membrane
• Two H+ are from the matrix, two from QH2
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Electron transfer and proton Electron transfer and proton flow in Complex IIIflow in Complex III
• Four H+ are translocated, two from the matrix and two from QH2
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Proposed Q cycle.Proposed Q cycle.
• Proposal for electron movement between between QH2 and cytochrome c in Complex III
• One e- transferred to cytochrome c via the Fe-S protein
• Second e- transferred to cytochrome b then Q
• Three forms of Q are involved: QH2, Q and the
semiquinone anion .Q-
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(a) Ubiquinone, (a) Ubiquinone, (b) Plastoquinone(b) Plastoquinone
• Hydrophobic tail of each is composed of 6 to 10 five-carbon isoprenoid units
• The isoprenoid chain allows these quinones to dissolve in lipid membranes
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• Three oxidation states of ubiquinone
• Ubiquinone is reduced in two one-electron steps via a semiquinone free radical intermediate. Reactive center is shown in red.
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Oxidative Phosphorylation and
Q Cycle(animation)
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Electron transfer and proton flow Electron transfer and proton flow in Complex IVin Complex IV
• Iron atoms (hemes of cyt a) and copper atoms are both reduced and oxidized as electrons flow
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Complex IV contributes to the Complex IV contributes to the proton gradientproton gradient
Net effect is transfer of four H+ for each pair of e-
O2 + 4 e- + 4H+ 2 H2O
1. Proton translocation of 2 H+ for each pair of electrons transferred (each O atom reduced)
2. Formation of H2O removes 2H+ from the matrix (contributes to p even though no proton translocation occurs)
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Proposed mechanism for reduction of Proposed mechanism for reduction of molecular oxygen by cytochrome oxidasemolecular oxygen by cytochrome oxidase
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Complex V: ATP SynthaseComplex V: ATP Synthase• F0F1 ATP Synthase uses the proton gradient
energy for the synthesis of ATP
• An F-type ATPase which generates ATP
• Composed of a “knob-and-stalk” structure
• F1 (knob) contains the catalytic subunits
• F0 (stalk) has a proton channel which spans the membrane.
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Knob-and-stalk structureKnob-and-stalk structure of ATP synthase of ATP synthase
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ATP Synthase
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ATP Synthase componentsATP Synthase components
• Passage of protons through the Fo (stalk) into the matrix is coupled to ATP formation
• Estimated passage of 3 H+ / ATP synthesized
• Fo is sensitive to oligomycin, an antibiotic that binds in the channel and blocks H+ passage, thereby inhibiting ATP synthesis
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Structure of ATP synthaseStructure of ATP synthase
• F1 knobs: inner face of the inner mitochondrial membrane (subunit composition: 33
• Fo subunit composition: a1b2c9-12
(c subunits form cylindrical, membrane-bound base)
• 33 oligomer of F1 is connected to c subunits by a multisubunit stalk of and chains
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ATPase mechanismATPase mechanism
The a-b-33 unit is the “stator” (the Fo channel is attached to 33 by the a-b- arm)
Passage of protons through the Fo channel causes the rotor to spin in one direction and the stator to spin in the opposite direction
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Experimental observation of ATP Experimental observation of ATP synthase rotationsynthase rotation
• Fluorescent protein arm (actin) attached to subunits
• subunits bound to a glass plate
• Arm seen rotating when MgATP added
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Active Transport of ATP, ADP and PActive Transport of ATP, ADP and Pii
Across the Mitochondrial MembraneAcross the Mitochondrial Membrane
• ATP is synthesized in the mitochondrial matrix
• ATP must be transported to the cytosol, and ADP and Pi must enter the matrix
• ADP/ATP carrier exchanges mitochondrial ATP4-
for cytosolic ADP3-
• The exchange causes a net loss of -1 in the matrix (draws some energy from the H+ gradient)
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Transport of ATP, ADP and PTransport of ATP, ADP and Pii across across
the inner mitochondrial membranethe inner mitochondrial membrane• Adenine nucleotide translocase: unidirectional
exchange of ATP for ADP (antiport)
• Symport of Pi and H+ is electroneutral
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The P:O RatioThe P:O Ratiomolecules of ADP phosphorylated
P:O ratio = -----------------------------------------atoms of oxygen reduced
• Translocation of 3H+ required by ATP synthase for each ATP produced
• 1 H+ needed for transport of Pi, ADP and ATP
• Net: 4 H+ transported for each ATP synthesized
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Calculation of the P:O ratioCalculation of the P:O ratioComplex I III IV
#H+ translocated/2e- 4 4 2
Since 4 H+ are required for each ATP synthesized:
For NADH: 10 H+ translocated / O (2e-)
P/O = (10 H+/ 4 H+) = 2.5 ATP/O
For succinate substrate = 6 H+/ O (2e-)
P/O = (6 H+/ 4 H+) = 1.5 ATP/O
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UncouplersUncouplers
• Uncouplers stimulate the oxidation of substrates in the absence of ADP
• Uncouplers are lipid-soluble weak acids
• Both acidic and basic forms can cross the inner mitochondrial membrane
• Uncouplers deplete any proton gradient by transporting protons across the membrane
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2,4-Dinitrophenol: an uncoupler2,4-Dinitrophenol: an uncoupler
• Because the negative charge is delocalized over the ring, both the acid and base forms of DNP are hydrophobic enough to dissolve in the membrane