Oxidative Phosphorylation

Coenzyme Oxidation and Redox Potentials:

NADH_(reductant) + H⁺ + 1/2O_2(oxidant) → NAD⁺ + H₂O

• Oxidation

  • NAD⁺ + 2H⁺ + 2e^- → NADH + H⁺, E⁰ = -0.32V.

• Reduction

  • 1/2O₂ + 2H⁺ + 2e^- → H₂O, E⁰ = 0.816V

ΔE⁰ = E⁰_{(reduction/acceptor)} - E⁰_{(oxidation/donor)}

• ΔE⁰ = 0.816 -(-0.32) = 1.136V.

The nernst equation shows that ΔG⁰ = -nFE⁰.

• ΔG⁰ = -(2) x (96.485kJ/Vmol) = -219.21kJ/mol

A reaction with a net positive ΔE⁰ yields a negative ΔG⁰ which results in a spontaneous exergonic reaction.

Molecules along the ETC have reduction potentials between the values of the NAD⁺/NADH couple and the O₂/H₂O couple.

• So the electrons move down the energy scale towards progressively more positive reduction potentials.

OXPHOS-ETC Complexes:

There are four protein complexes in the ETC:

• NADH-Coenzyme Q Reductase (I)

• Succinate-Coenzyme Q Reductase (II)

• Coenzyme Q-Cytochrome C Reductase (III)

• Cytochrome C Oxidase (IV)

+ ATP Synthase

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Molecular Components of Complexes:

• Flavoproteins:

  • Tightly bound FMN or FAD (prosthetic groups) may participate in 1 or 2 e^- transfer events.

• Coenzyme Q (Ubiquinone; CoQ; UQ):

  • 1 or 2 e^- transfer reactions.

• Cytochromes (b, c, c₁, a and a₃):

  • Haem (prosthetic groups) are 1e^- transfer agents (i.e. Fe³⁺ → Fe²⁺).

• Fe-S Proteins:

  • 1e^- transfer (Fe²⁺ and Fe³⁺ states).

• Protein-bound Cu²⁺:

  • 1e^- transfer sites (Cu⁺ → Cu²⁺).

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Overview of the Complexes:

• Complex I:

  • Accepts 2e^- from NADH (links between glycolysis, TCA, B-oxidation and ETC).

• Complex II:

  • Includes succinates dehydrogenase (links between TCA and ETC).

    • Entry point for FADH₂e^- from TCA.

Complexes I and II produce a common product (reduced coenzyme Q (UQH₂)) which is a substrate for complex III.

• Complex III:

  • Oxidises UQH₂ while reducing cytochrome c (substrate for complex IV).

• Complex IV:

  • Reduces molecular oxygen.

Coenzyme Q:

Coenzyme Q is a mobile electron carrier.

• It is highly hydrophobic.

• Diffuses freely in the hydrophobic core of the inner-mitochondrial membrane.

• Can take part in 1e^- or 2e^- reactions.

Complex I: NADH-Coenzyme Q Reductase:

• ~900kDa

• More than 30 polypeptide chains.

• 1 molecule of FMN.

• As many as 7 Fe-S clusters.

  • Containing a total of 20-26 iron atoms.

1. Step 1:

• NADH binds to complex I on the matrix side of the IMM.

  • Transfer of 2e^- from the NADH to FMN:

    • NADH + [FMN] + H⁺ → [FMNH₂] + NAD⁺.

2. Step 2:

• Transfer of 2e^- from the [FMNH₂] → series of Fe-S proteins.

3. Step 3:

• 2e^- are transferred from Fe-S clusters to coenzyme Q.

Some of the energy liberated by the flow of e^- through this complex is used in a ‘coupled’ process to drive protons across the membrane.

• As 2e^- flow from NADH to Co-Q, 4H⁺ are pumped out across IMM.

Complex II: Succinate-Coenzyme Q Reductase:

• The only TCA enzyme that is an integral membrane protein in the IMM.

• ~100-140kDa.

• FAD is covalently bound to a histidine residue in a 68kDa flavoprotein.

• 3 Fe-S clusters in 29kDa protein.

• 2 small subunits, with haem b that binds UQ.

1. Step 1:

• Succinate → Fumarate (reduction of bound FAD to FADH₂).

2. Step 2:

• FADH₂ transfers e^- immediately to Fe-S centres → UQH₂.

The small ΔG⁰ (-5.6kJmol^-1) is insufficient to transport H⁺ across the IMM.

Cytochrome C:

It is a mobile e^- carrier that is H₂O soluble.

• It is globular, meaning that the haem group lies in the centre of the protein.

• Associates along the membrane surface in its reduced state.

• Carries e^- to the 4th complex (cytochrome c oxidase).

Complex III: Coenzyme Q-Cytochrome c Reductase:

It is a dimer consisting of 11 proteins, 248kDa per monomer.

• Fe-S Rieske protein (iron sulphur protein (ISP)).

• 3 Different Cytochromes:

  • Cyt b (2 haem groups, b_L and b_H).

  • Cyt c₁.

  • Cyt c (loosely associated).

Passage of e^- through the complex is accompanied by proton transport across the IMM, which is a complicated 2 step Q cycle*, with 2 QH₂ molecules.

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*Q Cycles:

The Q cycle takes place in Complex III. In the first half of the cycle, two electrons of a bound QH₂ are transferred.

• One to cytochrome c.

• One to a bound Q in the second binding site to form the semiquinone radical anion Q.

The newly formed Q dissociates and enters the Q pool.

• In the second half of the cycle, a second QH₂ also gives up its electrons to Complex III.

  • One to a second molecule of cytochrome c.

  • One to reduce Q to QH₂.

This second electron transfer results in the uptake of two protons from the matrix.

Complex IV: Cytochrome c Oxidase:

Consists of 13 subunits, with a size of 204kDa.

• 2 Cu centres (2 x Cu_A and 1 x Cu_B), which associate with cytochromes a and a₃ respectively.

• Centres contained within core subunits I, II and III, which induce redox centres and a proton channel.

Reduction of O₂ requires the passage of 4e^- through this complex.

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Some of the unused free energy released on e^- transfer from cytochrome c to O₂ is harnessed by pumping 4H⁺ out through a proton channel.

• 4Cytc_red + 8H⁺_insitu + O₂ → 4Cytc_oxi + 2H₂O + 4H⁺_out

Complex I, III and IV Drive H⁺ Across the IMM:

• Stores the energy of electron transport in an electrochemical membrane potential.

  • H+ is driven out of the matrix, causing pH to rise, and the matrix becomes negatively charged.

  • Both charge and concentration difference tend to attract H⁺ back into the matrix.

    

ATP Synthase:

It is a complex that carries out ATP synthesis.

• ATP synthase/F₁F₀-ATPase.

It harnesses H⁺ flux to drive ATP synthesis.

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ATP Synthase Structure:

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ATP Synthesis is driven by Conformational Changes:

1. Translocation of H⁺:

• Carried out by F₀, causing rotation of the c complex and rotation of the γ/ε stalk.

2. Catalysis of ATP Synthesis:

• Carried out by F₁, causing conformational changes in β-subunits which alter binding affinity for ATP/ADP, and stabilise ATP.

  • Energy drives ATP dissociation.

3. Coupling of H⁺ Gradient Dissipation with ATP Synthesis:

• H⁺ leakage through F₀ drives the molecular rotor (γ/ε), the interactions of γ with the β-subunit drive conformational change.

The Electron Transport Chain:

The synthesis of ATP results in the translocation of 3H⁺ from the IMS to the matrix via F₀.

• 4H⁺ (1H⁺ to bring substrate P_i) are transported to the matrix per ATP synthesised and moved to the cytosol.

  • Therefore 25% of the energy derived from ETC + OxiPhos is consumed as the electrochemical energy devoted to mitochondrial ATP-ADP transport.

Phosphorylation and Oxidation are Tightly Coupled:

• P/O Ratio:

  • The ratio of phosphate incorporated into ATP to oxygen atoms reduced to water.

    • It is a measure of the efficiency of coupling of phosphorylation to oxidation, and in turn is a measure of ATP production efficiency.

10H⁺ are transported out of the matrix per 2e^- passed from NADH → O₂.

4H⁺ are transported to the matrix per ATP generated.

• P/O ratio for NADH = 10/4 = 2.5

• P/O ratio for FADH₂ = 6/4 = 1.5