Oxidative Phosphorylation

• Outer membrane


  • Highly impermeable
  • Porins
• Inner membrane


  • Highly impermeable
  • Large surface area due to the invaginations of the membrane
  • Mitochondrial pyruvate carrier (MPC)
• There is no specific mitochondrial NADH transporter hence the cytosolic pool of NADH is different from the matrix mitochondrial pool
• Porins allow NADH access to the intermitochondrial space for small molecules
• FADH2 is made on the inner membrane
• Succinate dehydrogenase sits in the inner membrane and is also a component of the electron transport chain
• Malate-Aspartate Shuttle


  • Two specific inner mitochondrial transporters – malate and aspartate
  • Results in the electrons and protons from the cytoplasmic NADH being transferred to the NAD+ in the mitochondrial matrix
  • Any NADH made using the malate-aspartate shuttle will result in the production of 3 ATP per NADH
  • Occurs in tissues where the energy requirements is relatively low such as the liver
• Glycerol-Phosphate Shuttle


  • Used in metabolically active tissue such as neurons and muscle
  • There are two glycerol-3-P dehydrogenase enzymes (cytosolic and the other mitochondrial bound with the active site on the intermembraneous side)
  • The glycerol-3-phosphate is produced in the cytoplasm and can easily move into the intermembranous space of the mitochondria
  • The DHAP can pass back into the cytoplasm and be used in glycolysis
  • The one less ATP ensures that this reaction is not able to reverse as it decouples the matrix NADH from the cytoplasmic NADH
  • This oxidation/reduction cycle produced FADH2 hence using this glycerol-phosphate shuttle produces a total of 2 ATP per NADH
• Energy balance sheet

  • 2 ATPs used to prime glycolysis
  • 4 ATPs produced from the glycolytic pathway as a result of substrate level phosphorylation
  • 2 NADHs generated (equivalent to 3 ATPs per NADH oxidised by there electron transport chain using the malate-aspartate shuttle)
  • 8 ATPs net gained from the conversion of glucose to pyruvate and the re-oxidation of the 2 NADHs produced
• Balance sheet for the complete oxidation of glucose

  • Glycolysis generates 8 ATP and the TCA cycle generates 30 ATP for 2 pyruvates entering the TCA cycle using only the malate-aspartate shuttle
  • Glycolysis generates 6 ATP and the TCA cycle generates 30 ATP for 2 pyruvates entering the TCA cycle using only the glycerol-phosphate shuttle
  • Total net ATP ranges from 36 to 38
• Oxidation reactions are exergonic
• Coupling electron transport to ATP synthesis


  • The energy generated as electrons are passed from NADH/FADH2 to oxygen but this processes is not directly coupled to ATP synthesis
  • The energy is couple to the transport of protons (H+) from the mitochondrial matrix to the intermembranous space
  • This generates an electrochemical gradient across the highly impermeable inner membrane
  • The electrochemical gradient consists of two parts, the chemical gradient, or difference in solute concentration across a membrane, and the electrical gradient, or difference in charge across a membrane
• Oxidative phosphorylation is not directly coupled to ATP synthesis
• Electrochemical gradient


  Consists of an electrical (charge separation) and a chemical (concentration gradient) component
• Oxidation of NADH - Complex I


  1. Complex I oxidises NADH and transfers 2 electrons to ubiquinone

  2. This is accompanied by the transfer of approximately 4 protons across the inner mitochondrial membrane

  3. Reduced ubiquinone (ubiquinol) moves throughout the inner membrane

  4. Ubiquinol delivers its electrons to complex III
• Oxidation of NADH - Complex III


  1. Complex III transfers two electrons between two mobile electron carriers

  2. It receives them from ubiquinol and transfers them to soluble cytochrome-c

  3. 1 electron is transferred to 1 cytochrome-c with the transfer of approx. 2H+

  4. The second electron is first stored internally and then transferred to a second cytochrome c which also results in two additional H+ being transferred from matrix to IMS

  5. Thus there are a total of 4H+ transferred, 2 from ubiquinol and 2 from the matrix
• Oxidation of NADH - Complex IV


  1. Complex IV transfers the two electrons from the two cytochrome-c proteins to the mitochondrial matrix

  2. This is accompanied by the transfer of 2H+ to the intermembranous space and reducing oxygen on the matrix side to produce H2O

  3. Hence, the oxidation of one NADH results in the transfer of approx. 10 H+ (4 from Complex I, 4 from Complex III and 2 from Complex IV) to the intermembraneous space
• Oxidation of FADH2 - Complex II, III and IV


  1. Complex II converts succinate to fumarate, reducing FAD to FADH2

  2. For the oxidation of FADH2, complex I is not utilised hence fewer protons are transferred across the inner membrane

  3. The oxidation of FADH2 only transfers 6 H+ (4 from Complex III and 2 from Complex IV) to the IMS
• ATP synthase

  • The central rotor stalk (gamma subunit) has a protrusion, and as this rotates it moves the alpha and beta domains
  • The head is a heterohexamer, made up of 3α and 3β subunits
  • Roughly, 3 H+ are required per ATP
• Rotation drives ATP synthesis

  1. The return of the H+s causes rotation of the spindle of the ATP synthase (rotor)

  2. The rotor is asymmetric and causes conformational changes in the head group subunits that make up the stator

  3. These conformational changes in the stator are involved in driving the synthesis of ATP from ADP and Pi
• Complex I entry: 1 NADH ~ 10 H+ transferred to IMS, with ~ 3 H+ per ATP hence ~ 3 ATP/NADH
• Complex II entry: 1 FADH2 ~ 6 H+ transferred to IMS, with ~ 3 H+ per ATP hence ~ 2 ATP/NADH
• If there is no ADP

  • ATP cannot be exchanged from the mitochondrial matrix
  • H+ cannot return to the mitochondrial matrix
  • The rate of electron transport will stop
  • ATP will no longer be produced
• When there is a cytoplasmic demand for energy

  • ADP level rises
  • ADP enters the matrix and ATP leaves
  • ADP can now be phosphorylated by the ATP synthase using the proton motive force
  • The proton motive force is reduced
  • The electron transport chain starts up again to renew the proton motive force
• Succinate dehydrogenase links the TCA cycle with oxidative phosphorylation
• What is the evidence suggesting that mitochondria were once free-living prokaryotes?

  Some of the evidence includes mitochondrial DNA, which is similar to bacterial DNA, and the double membrane structure of mitochondria, which is consistent with the engulfing mechanism of endosymbiosis
• How much ATP is generated from the complete oxidation of glucose including glycolysis and the Krebs cycle?


  From the complete oxidation of glucose, 8 ATP are generated from glycolysis, and 30 ATP from the Krebs cycle, resulting in a total of 38 ATP
• What is the role of the malate-aspartate shuttle?


  The malate-aspartate shuttle transfers electrons from cytoplasmic NADH to mitochondrial NAD+, facilitating the movement of electrons into the mitochondrial matrix for ATP production
• What is the difference in ATP yield between the malate-aspartate shuttle and the glycerol-phosphate shuttle?

  The malate-aspartate shuttle yields 3 ATP per NADH, while the glycerol-phosphate shuttle yields 2 ATP per NADH
• How does succinate dehydrogenase link the TCA cycle to the electron transport chain?


  Succinate dehydrogenase, also known as complex II, participates in both the TCA cycle and the electron transport chain by converting succinate to fumarate and reducing FAD to FADH2, which then enters the electron transport chain
• What is the purpose of oxidative phosphorylation in mitochondria?


  Oxidative phosphorylation's primary purpose is to generate ATP from ADP and inorganic phosphate (Pi), using the energy released by the oxidation of NADH and FADH2 in the electron transport chain
• How is the electrochemical gradient across the mitochondrial inner membrane used in ATP synthesis?

  The electrochemical gradient, consisting of both a proton gradient and an electrical charge difference, powers ATP synthase to synthesize ATP from ADP and Pi
• What stops the ATP synthase activity when there is no ADP available in the mitochondrial matrix?

  Without ADP to convert to ATP, ATP synthase activity halts because there's no substrate for phosphorylation, leading to an accumulation of the proton gradient to a point where it no longer drives ATP synthesis