Oxidative phosphorylation: Electron transport chain and ATP synthesis

August 8, 2020 Gaurab Karki Biochemistry 0




Oxidative phosphorylation: Electron transport chain and ATP synthesis
Oxidative phosphorylation: Electron transport chain and ATP synthesis

Oxidative phosphorylation:

  • Reducing equivalent NADH, FADH2 generated during glycolysis and the link between glycolysis and Kreb’s cycle are used to synthesize ATP by a process called oxidative phosphorylation (OP).
  • Oxidative phosphorylation involves two components-
    • Electron transport chain
    • ATP synthase.
  • The flow of electrons from the reducing equivalence across the electron transport chain generates proton motive force (PMF).
  • The energy stored in proton motive force is used to drive the synthesis of ATP.
  • ATP synthase utilizes this proton motive force to drive the synthesis of ATP.

Electron transport chain:

  • Electron transport chain consists of the series of electron carriers arranged asymmetrically in the membrane.
  • The membrane may be either cytoplasmic membrane as in the case of bacteria or inner mitochondrial membrane as in case of eukaryotes.
  • The electron carriers are sequentially arranged and get reduced as they accept electron from the previous carrier and oxidized as they pass electron to the succeeding carrier.
  • The different electron carriers are:
    • NADH dehydrogenase
    • Flavoproteins (FMN and FAD)
    • Ubiquinone
    • Iron sulfur (Fe) center
    • Cytochrome
fig. Different electron carriers

1. NADH dehydrogenase:

  • Two types of NAD dependent dehydrogenase can feed electron transport chain.
  • They are NADH and NADPH.
  • NADPH is less common as it is involved in anabolic reactions (biosynthesis).
  • NADH dehydrogenase removes two hydrogen atoms from the substrate and donates the hydride ion (H–) to NAD+ forming NADH and H+ is released in the solution.
  • NAD+ accepts two e– and two protons from the substrate during catabolic reaction and transfers to the electron transport chain.
  • NAD+ is then reduced to NADH+ H+.
  • Reduced NADH+ H+ transfers its e– and proton to FMN which in turn is reduced to FMNH2.
    • AH2+ NAD+   <——————–>A + NADH + H+
    • (Reduced substrate)                 (oxidized substrate)
    • NADH + H+ + FMN  <———–> FMNH2+ NAD+

2. Flavoproteins:

  • Flavoproteins are derived from Vitamin B2 (Riboflavin).
  • These are the protein containing FMN and FAD as the prosthetic group which may be covalently bound with the protein.
  • They are capable of accepting electrons and protons but can only donate electrons.
  • The protons are expelled outside the membrane.
  • FMN accept electron and proton from NADH and get reduced to FMNH2 which in turn channel only e– through to ubiquinone.
  • FAD is the component of succinate dehydrogenase complex.
  • It accepts two electron and two protons from succinate and gets reduced to FADH2, in the process succinate is converted to fumarate.
  • FADH2 channels its electron only to FeS center through ubiquinone.
  • Succinate+ FAD ____________________> Fumarate + FADH2

3. Ubiquinone:

  • Ubiquinone are omnipresent in nature.
  • These are similar in structure and property with Vitamin K.
  • In plants, these are found as plastoquinone and in bacteria, these are found as menaquinone.
  • These are lipid soluble (hydrophobic) and can diffuse across the membrane and channel electrons between carriers.
  • Ubiquinone can accept electrons as well as protons but transfer only electrons.
  • They accept electron from complex 1 and 2.
  • They can accept one e– and get converted into semiquinone or two e–s to from quinone.

4. FeS center:

  • These are non-heme Fe (iron) containing proteins in which the Fe-atom is covalently bonded to Sulphur of cysteine present in the protein and to the free Sulphur atoms.
  • Less commonly found FeS centers known as Reiske iron sulphur centers have iron bonded to Histidine residue of the proteins.
  • There are different types of iron Sulphur center, simplest type consists of an iron atom, another type known as 2Fe-2S (Fe2S2) and the third one (most commonly found) is 4Fe-4S (Fe4-S4) and comprises the ferredoxin.
  • FeS center consists of Fe-atoms which can interconnect between ferrous and ferric form as they accept and donate electrons respectively.
  • They are capable of receiving and donating electrons only.
  • They form the components of all four complexes.

5. Cytochromes:

  • Cytochromes are the proteins with characteristic absorption of visible lights due to the presence of heme containing Fe as co-factor.
  • There are three different types of cytochrome a, b and c.
  • Cytochrome a and b are tightly but not covalently linked with their proteins whereas cytochrome c is covalently bonded with its protein through cysteine.
  • Cytochrome ‘a’ has the maximum absorption spectra at 600nm.
  • Cytochrome ‘b’ has maximum absorption spectra at 560nm and cytochrome ‘c’ has maximum absorption spectra at 550nm.
  • Cytochromes are capable of accepting and transferring only one e– at a time during which the Fe– atoms interconvert between ferrous and ferric.
  • Cytochrome- Fe2+ <————> Cytochrome- Fe3+ + e–
  • Cytochromes are arranged in the order cytochrome ‘b’, cytochrome c1, cytochrome ‘c’ and cytochrome a/a3.
  • a/a3 is also known as cytochrome oxidase.

Arrangement of five electron carriers in the form of four respiratory enzyme complex

  • The five electrons carriers are arranged in the form of four complexes.
    • Complex I: NADH Quinone oxidoreductase complex (NADH to Quinone)
      Note: NADH——->FMN——> FeS—–> Q
    • Complex II: Succinate dehydrogenase complex (Succinate to Quinone)
      Note: Succinate——> FAD—–> FeS—-> Q
    • Complex III: cytochrome bc1 (Ubiquinone to cytochrome c)
      Note: UQ2——> cyt bc1—->cyt c
    • Complex IV: Cytochrome oxidase (cytc to O2)
      Note: cyt c—-> cyt a—–> cyt a3—-> O2

Complex I: NADH dehydrogenase complex

  • This complex is also known as NADH dehydrogenase complex, consists of 42 different polypeptides, including FMN containing flavoprotein and at least six FeS centers.
  • Complex I is ‘L’ shaped with its one arm in the membrane and another arm extending towards the matrix.
  • During catabolic reaction, NAD+ is reduced to NADH+ H+ and this NADH + H+ feeds electrons and protons at the point of origin in the ETC.
  • Both e– and protons are transported to FMN which is then reduced to FMNH2.
  • FMNH2 transfers only e– to FeS center whereas protons are extruded outside the membrane (intermembrane space), in the process FMNH2 is oxidized back to FMN.
  • Electrons flow through FeS centers which alternate between reduced (Fe2+) and oxidized (Fe3+) froms.
  • Electrons are finally transferred to ubiquinone, which along with protons obtained by the hydrolysis of water in the matrix site of the membrane is reduced to UQH2.

Complex II: Succinate dehydrogenase complex.

  • Complex II is also known as succinate dehydrogenase complex.
  • Succinate dehydrogenase complex is located towards the matrix side of the membrane.
  • Succinate is oxidized to fumarate as it transfers two e–s and two protons to FAD.
  • FAD is reduced to FADH2.
  • FAD transfers only electrons through FeS center to quinone.
  • Quinone (Q) in presence of protons is reduced to QH2.
  • Complex II consists of covalently linked FAD containing flavoprotein and two FeS centers.

Complex III: Cytochrome bc1

  • Ubiquinone are hydrophobic, lipid soluble molecules capable of diffusing across the membrane.
  • Because of this property, ubiquinones can channel electrons between less soluble electron carriers.
  • Electrons are channeled from complex I and complex II to cytochrome bc1.
  • The figure shows the stoichiometry for two ubiquinone (UQH2).
  • Ubiquinones undergo two rounds of oxidation, one towards the enzyme site on the inner membrane site of the membrane where two electrons are transferred across cyt c1 to cyt c.
  • Another oxidation occurs towards the site of membrane containing cyt b where again 2 electrons are passed to cyt bc and cyt bH.
  • During these two oxidation reactions, four protons are expelled outside the membrane and 2UQH2 is oxidized to 2UQ.
  • One of the UQ diffuse towards the matrix site of the membrane where it receives two electrons flowing through cytochrome b1.
  • This UQ along with two protons obtained from the hydrolysis of water in the matrix site of the membrane is reduced to UQH2, thus completing the Q-cycle.
fig. Oxidation of Ubiquinone

Complex IV: Cytochrome Oxidase

  • It is also called as cytochrome oxidase.
  • Cytochrome c undergoes oxidation in the side of the membrane facing the intermembrane space and O2 is reduced in the matrix side of the membrane to H2O.
  • Complex IV consists of iron containing heme-a and heme-a3.
  • Along with iron atoms, cytochrome oxidase also consists of Cu A and Cu B.
  • Cu A is closely but not intimately associated with heme ‘a’ and Cu B is intimately associated with heme a3.
  • Electrons from cytochrome c flows to Cu A and then to heme ‘a’ and then to heme a3 and then to Cu B and then finally to Oxygen.
    • Cytochrome c —> Cu A —–> Heme a—–> heme a3—->Cu B—> O2
  • The copper atoms interconvert between cuprous (reduced) and cupric (oxidized).
  • Electrons from Cu B and heme a3 is transferred to O2 forming O–-O– bridge.
  • Two more electrons are pass through O–-O– resulting in breakage of O–-O– bridge forming O2– and O2-.
  • Two protons are supplied from the matrix side forming OH– and OH–.
  • Now, addition of two more proton from matrix side resulting in formation of two molecule of water (2H2O).

ATP synthesis:

  • Chemiosmotic theory given by Peter Mitchell (1961) in the widely accepted mechanism of ATP generation.
  • According to this theory electron and proton channel into the membrane from the reducing equivalence flows through a series of electron carriers, electrons flow from NADH through FMN, Q, cytochrome and finally to O2.
  • However, proton as they flow through the membrane are extended at different position in the intermembrane space.
  • The extension of protons creates a slight positivity/acidity to the outerside of membrane.
  • Reduction of quinones and O2 to water requires protons which are provided by the hydrolysis of water in the matrix side of the membrane.
  • This results in accumulation of hydroxyl ion in the inner (matrix) side of membrane resulting in slight negativity/alkalinity in the inner side of the membrane.
  • This creates a charge difference between outer side of the membrane, and inner side of membrane which energizes the membrane.
  • This is electrochemical potential, and this potential along with the pH gradient generates the proton motive force (PMF).
  • This proton motive force tends to drive the proteins through ATP synthase in to the inner side of the membrane, the consequence of which is ATP production.
  • ATP synthase consists of two components, transmembrane ion conducting subunit called Fo and cytoplasmic multiprotein subunit called F1 which is responsible for ATP production.
  • F1 catalyzes the reversible reaction in which ADP is phosphorylated to ATP.
    • ADP + Pi <————->ATP
  • Proton motive force driven H+ through Fo causes the rotation of C-protein of the subunit.
  • Rotation of c generates torque.
  • This torque is transmitted through  gamma (γ) and epsilon (ε) subunit to β-subunit of F1 resulting in its conformational change.
  • This conformational change in β-subunit allows binding of ADP with inorganic phosphate (Pi).
  • Binding of ADP and Pi results in production of ATP and β-subunit original conformation is regained.

Oxidative phosphorylation: Electron transport chain and ATP synthesis