Oxidative Phosphorylation

Oxidative Phosphorylation

Oxidative Phosphorylation


       At the end of this lecture, student will be able to

      Explain oxidative phosphorylation

      Describe the mechanism of oxidative phosphorylation

      Discuss the uncouplers of oxidative phosphorylation

      Explain substrate level phosphorylation

Oxidative Phosphorylation

       The process of synthesizing ATP from ADP and Pi coupled with the ETC is known as oxidative phosphorylation

       Complex V of the inner mitochondrial membrane is the site of oxidative phosphorylation

Sites of oxidative phosphorylation in ETC

There are three reactions in the ETC that result in the synthesis of 3 ATP molecules

                1. Oxidation of FMNH2 by coenzyme Q

                2. Oxidation of cytochrome b by cytochrome c1

                3. Cytochrome oxidase reaction

       Each one of the above reactions represents a coupling site for ATP production

Energetic of oxidative phosphorylation

       The transport of electrons from redox pair NAD+/NADH (Eo= -0.32) to finally the redox pair ½ O2 (Eo= + 0.82) may be simplified and represented in the following equation

       The redox potential difference between these two redox pairs is 1.14V, which is equivalent to an energy 52 Cal/mol

       3 ATP are synthesized in the ETC when NADH is oxidized which equals to 21.9 Cal (each ATP= 7.3 Cal)

       Efficiency of energy conservation is calculated as

       Hence, when NADH is oxidized, about 42% of energy is trapped in the form of 3 ATP and the remaining is lost as heat

Mechnism of Oxidative phosphorylation

       Several hypotheses have been put forth to explain the process of oxidative phosphorylation, among them most important is chemical coupling and chemiosmotic hypothesis

Chemical coupling hypothesis

       Edward Slater in 1953

       According to this, during electron transfer in respiratory chain, a series of phosphorylated high-energy
intermediates are first produced which are utilized for the synthesis of ATP

       These reactions are analogous to substrate level phosphorylation that occurs in glycolysis or TCA cycle

       However, this hypothesis lacks experimental evidence, since all attempts, so far, to isolate any one of the high-energy intermediates have not been successful

Chemi osmotic hypothesis

       By Peter Mitchell in 1961 & widely accepted

       lt explains how the transport of electrons through the respiratory chain is effectively utilized to produce ATP from ADP + Pi

       Proton gradient: The mitochondrial membrane is impermeable to protons (H+) and hydroxyl ions (OH-)

       The transport of electrons through ETC is coupled with the translocation of protons (H+) across the inner mitochondrial membrane from the matrix to the intermembrane space, where pumping of protons results in an electrochemical or proton gradient

       This is due to the accumulation of more H+ ions (low pH) on the outer side of the inner mitochondrial membrane than the inner side

       The proton gradient developed due to the electron flow in the respiratory chain is sufficient to result in the synthesis of ATP from ADP and Pi

Rotary and motor model

       Paul Boyer in 1964

       Also known as Boyer hypothesis / Rotary model/engine driving model / binding change model

       There is a conformational change in the mitochondrial membrane proteins leads to the synthesis of ATP

       The enzyme ATP synthase is F1 -F0 complex present in complex V

       F0 subcomplex is composed of channel protein ‘C’ subunits to which F1-ATP synthase is attached

ATP synthase-Rotary and motor model

Mechanism of Oxidative phosphorylation

Mechanism of Oxidative phosphorylation

       F1- ATP synthase consists of a central γ subunit surrounded by alternating α & β subunits (α3 & β3)

       In response to the proton flux, the γ subunit physically rotates

       This induces conformational changes in β3 subunits that finally lead to the release of ATP

       According to the binding change mechanism, the 3 β subunits of F1-ATP synthase adopt different conformations

      One subunit has open (O) conformation

      Second has loose (L) conformation

      Third one has tight (T) conformation

       The substrates ADP and Pi bind to β subunit in L-conformation

       The L site changes to T conformation and this leads to synthesis of ATP

       The T site changes to O conformation and releases ATP

       The O site changes to L conformation which binds to ADP and Pi

       Cycle is repeated & 3 ATP generated for each revolution

       The enzyme ATP synthase acts as a proton driving motor and is an example of rotary catalysis

       Thus, ATP synthase is the world‘s smallest molecular motor

ATP synthase-Rotary and motor model

Enzyme system for ATP synthesis

       ATP synthase present in the complex V, utilizes the proton gradient for the synthesis of ATP

       This enzyme is also known as ATPase since it can hydrolyse ATP to ADP and Pi

       ATP synthase is a complex enzyme and consists of two functional subunits, namely F1 and F0

       Its structure is comparable with ‘lollipops‘

       The protons that accumulate on the intermembrane space re-enter the mitochondrial matrix leading to the synthesis of ATP

Inhibitors of oxidative phosphorylation


       There are certain compounds that can uncouple the electron transport from oxidative phosphorylation, Such compounds are called as uncouplers, which increase the permeability of inner mitochondrial membrane to protons (H+)

       The uncouplers allow oxidation of substrates without ATP formation

       2,4-dinitrophenol (DNP) is an uncoupler

        small lipophilic molecule

       DNP is a proton-carrier, which easily diffuse through the inner mitochondrial membrane

       It is used in people seeking to lose weight & discontinued as it produces hyperthermia and other side effects

       Other uncouplers include dinitrocresol, entachlorophenol, carbonylcyanide trifluoromethoxy phenylhydrazone (FCCP)

       The last compound (FCCP) is said to be 100 times more effective than dinitrophenol

       High dose of aspirin acts as uncoupler

       Certain physiological substances like thermogenin, thyroxine and long chain free fatty acids in higher concentration act as Physiological uncouplers

       Unconjugated bilirubin is also believed to act as an uncoupler

       Significance of uncoupling

       Maintenance of body temperature is particularly important in hairless animals, hibernating animals and the animals adapted to cold

       The presence of active brown adipose tissue in certain individuals is believed to protect them from becoming obese

       The excess calories consumed by these people are burnt and liberated as heat, instead of being stored as fat

       Thermogenin or uncoupling protein (UCP) is a natural uncoupler located in the inner mitochondrial membrane of brown adipose tissue & blocks the formation of ATP and liberates heat

       lonophores promote the transport of ions across biological membranes. All the uncouplers are proton ionophores

       Antibiotics like valinomycin and nigercin act as ionophores for K+ ions & are capable of dissipating proton gradient across the inner mitochondrial membrane and inhibit oxidative phosphorylation

Other inhibitors of oxidative phosphorylation are

       Oligomycin: prevents the mitochondrial oxidation as well as phosphorylation. lt binds with the enzyme ATP synthase and blocks the proton (H+) channels

       Atractyloside: This is a plant toxin and inhibits oxidative phosphorylation by an indirect mechanism

Substrate level phosphorylation

       ATP is directly synthesized during substrate oxidation in the metabolism

       E.g. Succinyl CoA is converted to succinate by succinate thiokinase, This is a substrate-level phosphorylation, where GTP is converted to ATP by the enzyme nucleoside diphosphate kinase


       The process of synthesizing ATP from ADP and Pi coupled with the ETC is known as oxidative phosphorylation

       3 sites of oxidative phosphorylation are oxidation of FMNH2 by coenzyme Q, oxidation of cytochrome b by cytochrome c1 & Cytochrome oxidase reaction

       Chemical coupling and chemiosmotic hypothesis is the mechanism of oxidative phosphorylation

       ATP synthase present in the complex V, utilizes the proton gradient for the synthesis of ATP

       DNP, dinitrocresol, pentachlorophenol, and FCCP are uncouplers of oxidative phosphorylation

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