Oxidative Phosphorylation
Objective
• 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
• 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
• Uncouplers
• 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
Summary
• 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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