Oxidative phosphorylation includes the coupling of the oxidation of NADH or FADH2 by the respiratory chain with the synthesis of ATP via a gradient of protons across the inner mitochondrial membrane. Substrate level phosphorylation describes the formation of ATP or GTP linked to exergonic chemical reactions in metabolic pathways that provide the energy phosphorylation. Examples discussed previously include phosphoglycerate kinase and pyruvate kinase in glycolysis, succinyl CoA synthetase in the citric acid cycle, and creatin phosphokinase. Reduction potential (Eo) defines how well one substance reduces another (donate electrons). The oxidants accept electrons from a reductant.

Reduction-oxidation (redox) couple describes the pair of molecules of which one is reduced and the other is oxidized. Examples of redox pairs include lactate - pyruvate, NADH- NAD+, and FADH 2 -FAD. ∆Eo is defined as the standard reduction potential difference between two half reactions similar to the ∆Go that defines the standard energy difference. The value ∆Eo must be positive for the reaction to be spontaneous.

Overall respiratory (electron transport) chain reaction: Reduction potential of the H 2 O/ ½ O 2 and NADH/NAD + redox pairs. Oxidant Reductant Eo ½ O 2 H 2 O 0.82 V NAD NADH V In this instance although the respiratory chain contains numerous components, however the overall chain involves donation of electrons by NADH to reduce oxygen to water. Consequently the potential difference is calculated as: ∆Eo = (0.82 V) - (-0.32 V) = V.

The overall reaction becomes: NADH + H + + ½ O 2 ---> NAD + + H 2 O The Δ G o of this reaction equal to -52 Kcal The Δ G o of this reaction equal to -52 Kcal In fact sufficient energy is released to make several molecules of ATP from ADP and phosphate. In fact sufficient energy is released to make several molecules of ATP from ADP and phosphate.

Respiratory chain

From the previous figure we can see that the decrease in free energy occurs through the transfer of a pair of electrons from each carrier to the next. From the previous figure we can see that the decrease in free energy occurs through the transfer of a pair of electrons from each carrier to the next. -We note that: -We note that: -There are 3 large drops and 2 small drops. -There are 3 large drops and 2 small drops. The 3 large drops are: The 3 large drops are: 1- from NAD to coenzyme Q 2- From Cyt b to Cyt C 3- from Cyt a to oxygen

Components of ETC Electron transport chain complexes Electron transport chain complexes Enzymes involved in cellular redox reactions. Enzymes involved in cellular redox reactions. Proteins involved in redox reactions. Proteins involved in redox reactions.

Complex I NADH/ Coenzyme Q

Pyridine-linked dehydrogenases Also known as NAD(P) – linked dehydrogenases Also known as NAD(P) – linked dehydrogenases These dehydrogenases use NAD or NADP as electron acceptor These dehydrogenases use NAD or NADP as electron acceptor Most of thes are specific to NAD Most of thes are specific to NAD e.g: lactate dehydrogenase e.g: lactate dehydrogenase isocitrate dehydrogenase isocitrate dehydrogenase α- ketoglutarate dehydrogenase α- ketoglutarate dehydrogenase

Other require NADP Other require NADP e.g : Glucose -6- phosphate dehydrogenase e.g : Glucose -6- phosphate dehydrogenase All pyridine-linked dehydrogenases catalyze reversible reactions except glucose-6-phosphate dehydrogenase All pyridine-linked dehydrogenases catalyze reversible reactions except glucose-6-phosphate dehydrogenase They catalyze mitochondrial, cytosolic oxidations of CHO, F.A and amino acids They catalyze mitochondrial, cytosolic oxidations of CHO, F.A and amino acids

Some are located only in the cytosole: Some are located only in the cytosole: e. g: glyceraldhyde-3- p dehydrogenase e. g: glyceraldhyde-3- p dehydrogenase glucose-6-P dehydrogenase glucose-6-P dehydrogenase Others are located only in mitochondria Others are located only in mitochondria e.g: Pyruvate dehydrogenase e.g: Pyruvate dehydrogenase  - hydroxybutyrate dehydrogenase  - hydroxybutyrate dehydrogenase Some in both locations: Some in both locations: e.g: malate dehydrogenase e.g: malate dehydrogenase isocitrate dehydrogenase isocitrate dehydrogenase

Complex II

Flavin-linked dehydrogenases Also known as flavoproteins. Also known as flavoproteins. They use FMN and FAD They use FMN and FAD e. g : Succinate dehydrogenase. e. g : Succinate dehydrogenase. fatty acyl dehydrogenase fatty acyl dehydrogenase

Coenzyme Q (ubiquinone)

Coenzyme Q exists in mitochondria in the oxidized quinone form under aerobic conditions and in the reduced quinol form under anaerobic conditions. Coenzyme Q exists in mitochondria in the oxidized quinone form under aerobic conditions and in the reduced quinol form under anaerobic conditions. Structure is similar to vitamin K and E. All are characterized by the presence of polyisoprenoid side chain: Structure is similar to vitamin K and E. All are characterized by the presence of polyisoprenoid side chain: [ CH 2 -CH=C-CH 3 -CH 2 ]n. [ CH 2 -CH=C-CH 3 -CH 2 ]n.

The ETC contains excess of Coenzyme Q. This is compatible with Q acting as a mobile components of the ETC that collects reducing equivalents from the more fixed flavoprotein complexes and pass them to cytochromes. The ETC contains excess of Coenzyme Q. This is compatible with Q acting as a mobile components of the ETC that collects reducing equivalents from the more fixed flavoprotein complexes and pass them to cytochromes.

Iron-Sulfur protein complex (F 4 S 4 )

Iron sulfur protein complex (F 4 S 4 ) Iron sulfur protein complex (F 4 S 4 ) S - acid labile sulfur S - acid labile sulfur Cys - cysteine residue Cys - cysteine residue Pr - Proline Pr - Proline It is associated with flavoproteins (metalloflavoproteins) and with cytochrome b. It is associated with flavoproteins (metalloflavoproteins) and with cytochrome b. The sulfur and iron is thaught to take part in the oxidation reduction mechanism between flavin and coenzyme Q which involves a single electron change. The sulfur and iron is thaught to take part in the oxidation reduction mechanism between flavin and coenzyme Q which involves a single electron change. The iron atom undergoing oxidation reduction between Fe 2+ and fe 3. + The iron atom undergoing oxidation reduction between Fe 2+ and fe 3. +

Complex III

Cytochromes These are iron- containing electron transferring proteins. These are iron- containing electron transferring proteins. They are heme proteins. They are heme proteins. 3 classes have been identified a,b and c 3 classes have been identified a,b and c Each cytochrome molecule in its ferric (Fe 3+ ) form accepts one electron and reduced to the ferrous state (Fe 2+ ). Each cytochrome molecule in its ferric (Fe 3+ ) form accepts one electron and reduced to the ferrous state (Fe 2+ ). In addition to iron, Cyt a 3 also contain 2 bound copper atom which undergo cupric (Cu 2+ ) to cuprous (Cu + ) redox changes during electron transfer. In addition to iron, Cyt a 3 also contain 2 bound copper atom which undergo cupric (Cu 2+ ) to cuprous (Cu + ) redox changes during electron transfer.

Oxidative phosphorylations As a pair of electrons moves down the respiratory chain, 3 molecules of ATP are formed formed from ADP and phosphate. As a pair of electrons moves down the respiratory chain, 3 molecules of ATP are formed formed from ADP and phosphate. Such phosphorylation in the respiratory chain is known as oxidative phosphorylation or respiratory chain phosphorylation, therefore the true equation of respiratory chain electron transport is not. Such phosphorylation in the respiratory chain is known as oxidative phosphorylation or respiratory chain phosphorylation, therefore the true equation of respiratory chain electron transport is not. NAD red + 3 ADP + 3 Pi + 2H+ + ½ O 2 NAD red + 3 ADP + 3 Pi + 2H+ + ½ O 2 NAD ox + 3ATP + H2O

Because formation of 3 moles of ATP requires input of at least 3 X 7.3 = 21.4 K cal Because formation of 3 moles of ATP requires input of at least 3 X 7.3 = 21.4 K cal And oxidation of NAD red delivers 52 Kcal, And oxidation of NAD red delivers 52 Kcal, So we can deduce that phosphorylation of 3 moles of ADP conserves (21.4/52) x 100= 42% of the total energy yield when one mole of reduced NAD is oxidized by oxygen. So we can deduce that phosphorylation of 3 moles of ADP conserves (21.4/52) x 100= 42% of the total energy yield when one mole of reduced NAD is oxidized by oxygen. The difference is conserved as heat to maintain our body temperatures. The difference is conserved as heat to maintain our body temperatures.

The transfer of electrons down the the respiratory chain is energetically favoured (Spontaneous) because: The transfer of electrons down the the respiratory chain is energetically favoured (Spontaneous) because: - NADH is a strong electron donor and - NADH is a strong electron donor and - molecular Oxygen is strong electron acceptor. - molecular Oxygen is strong electron acceptor.

Chemiosmotic theory of ATP synthesis The chemiosmotic hypothesis (Mitchell hypothesis) explain how the free energy generated by the transport of electrons by the ETC is used to produce ATP from ADP and Pi. The chemiosmotic hypothesis (Mitchell hypothesis) explain how the free energy generated by the transport of electrons by the ETC is used to produce ATP from ADP and Pi. Electron transport in the chain is coupled with proton transport (H + ) across the innermembrane from the matrix to the intermembrane space. Electron transport in the chain is coupled with proton transport (H + ) across the innermembrane from the matrix to the intermembrane space.

This will creates: This will creates: - an electrical gradient ( more + ve charges on the outside of the membrane than the inside). - an electrical gradient ( more + ve charges on the outside of the membrane than the inside). - and pH gradient (The outside of the membrane is at lower pH than the inside). - and pH gradient (The outside of the membrane is at lower pH than the inside). “ The energy generated by this proton gradient is enough to drive ATP synthesis ” “ The energy generated by this proton gradient is enough to drive ATP synthesis ”

ATP synthetase: ATPase (Complex V) This enzyme complex synthesizes ATP, utilizing the energy of the proton gradient (Proton motive force) generated by the electron transport chain. This enzyme complex synthesizes ATP, utilizing the energy of the proton gradient (Proton motive force) generated by the electron transport chain. The Chemiosmotic theory proposes that after proton have transferred to the cytosolic side of inner mitochondrial membrane, they can reenter the matrix by passing through the proton channel in the ATPase (F 0 ), resulting in the synthesis of ATP in (F 1 ) subunit. The Chemiosmotic theory proposes that after proton have transferred to the cytosolic side of inner mitochondrial membrane, they can reenter the matrix by passing through the proton channel in the ATPase (F 0 ), resulting in the synthesis of ATP in (F 1 ) subunit.

Uncouplers Electron transport and phosphorylation can be uncoupled by compounds that increase the permeability of the innermitochondrial membrane to protons in any place. Electron transport and phosphorylation can be uncoupled by compounds that increase the permeability of the innermitochondrial membrane to protons in any place. i.e Uncouplers causes electron transport to be proceed at a rapid rate without the establishing of proton gradient i.e Uncouplers causes electron transport to be proceed at a rapid rate without the establishing of proton gradient e.g : 2,4 dinitrophenol

The energy produced by the transport of electrons is released as heat rather than being used to synthesize ATP. The energy produced by the transport of electrons is released as heat rather than being used to synthesize ATP. In high doses, the drug aspirin uncouple oxidative phosphorylation. This explain the fever that accompanies toxic overdoses of these drugs. In high doses, the drug aspirin uncouple oxidative phosphorylation. This explain the fever that accompanies toxic overdoses of these drugs.

Electron transport inhibitors These compounds prevent the passage of electrons by binding to chain components, blocking the oxidation/reduction reaction These compounds prevent the passage of electrons by binding to chain components, blocking the oxidation/reduction reaction Inhibition of electron transport also inhibits ATP synthesis. Inhibition of electron transport also inhibits ATP synthesis. e.g: - Amytal and Rotenone block e- transport between FMN and Co Q. e.g: - Amytal and Rotenone block e- transport between FMN and Co Q. - Antimycin A blocks between Cyt b and Cyt c - Antimycin A blocks between Cyt b and Cyt c - Sodium azide blocks between Cyt a a3 and oxygen - Sodium azide blocks between Cyt a a3 and oxygen

Ionophores Ionophores are termed because of their ability to form complex with certain cations and facilitate their transport across the mitochondrial membrane. Ionophores are termed because of their ability to form complex with certain cations and facilitate their transport across the mitochondrial membrane. So ionophores are lipophilic So ionophores are lipophilic e. g: Valinomycin: allows penetration of K + across the mitochondrial membrane and then discharges the membrane potential between outside and the inside e. g: Valinomycin: allows penetration of K + across the mitochondrial membrane and then discharges the membrane potential between outside and the inside ( i.e: does not affect the pH potential). ( i.e: does not affect the pH potential). Nigericin: also acts as ionophore for K + but in exchange with H +. It therefore abolishes the pH gradient. Nigericin: also acts as ionophore for K + but in exchange with H +. It therefore abolishes the pH gradient.