associated with the functioning of the electron transport chain. This was studied by fragmentation of mitochondria. In the first fragmentation step, the outer membrane is removed by treatment with various detergents such as saponin, digitonin. The two particulate fractions that result are:
1. The outer membrane, either in the form of vesicles or completely solubilised.
2. The inner membrane and the mitochondrial matrix enzymes. This fraction is found to contain the enzymes of:
• The electron transport chain • Oxidative phosphorylation • The TCA cycle.
In oxidative phosphorylation ATP is produced by combining ADP and Pi with the energy generated by the flow of electrons from NADH to molecular oxygen in the electron transport chain. There are three sites in the respiratory chain where ATP is formed by oxidative phosphorylation. These sites have been proved by the free energy changes of the various redox couples. Since hydrolysis of ATP to ADP + Pi releases around 7.3 K. Cal/mole, the formation ATP from ADP + Pi requires a minimum of around 8 KCal/mole. The formation of ATP is therefore not possible at the sites where free energy released is less than 8 KCal/mole.
Whenever two systems or redox couple of the respiratory chain differ from each other by 0.22 volts in standard redox potential (E’o), the free energy is sufficient to form ATP.
Go= –n F Eo
Go= –2 × 23.06 × 0.22 = –10.15 KCal
Sites of ATP formation: There are three sites in the respiratory chain where ATP can be formed.
• Site I: This involves the transfer of electrons from NADH –CoQ. Obviously this step is omitted by succinic
dehydrogenase whose FADH2 prosthetic group transfers
its electrons directly to CoQ bypassing NADH. This step is blocked by piericidin, rotenone, amobarbital, certain drugs like chlorpromazine, guanethidine. • Site II: This involves the transfer of electrons from Cyt-
b–Cyt-c1. This step as well as the previous one is bypassed in oxidation of L-ascorbate whose electrons are directly transferred to Cyt-c. This step is blocked by BAL, Antimycin A, Hypoglycaemic drug like phenformin.
SECTION TWO
• Site III: Transfer of electrons from Cyt-a3 to molecular oxygen which is blocked by CO, CN, H2S, and azide.
Mechanism of Oxidative Phosphorylation
Three major proposals for the mechanism of oxidative phosphorylation have been considered. The synthesis of ATP is carried out by a molecular assembly in the inner mitochondrial membrane. This enzyme complex is called Mitochondrial ATPase or H+-ATPase. It is also called ATP- synthase.
Theories
The three hypothesis do make use of the information available on ATP synthase.
A. The chemical coupling hypothesis: This is developed from the concept of a high energy intermediate common to both electron transport and phosphorylation of ADP. However, such intermediate has not been identified so far.
B. The conformational coupling hypothesis: According to this hypothesis the mitochondrial cristae undergo conformational changes and these changes in archi- tecture of the mitochondrial cristae reflect the changes in the different components of the electron chain to one another. It is believed that these conformational change represents the formation of high energy state.
C. Chemiosmotic theory: This is the most accepted view of oxidative phosphorylation postulated by Peter Mitchell in 1961. Mitchell’s chemiosmotic theory postulates that the energy from oxidation of
components in the respiration chain is coupled to the translocation of hydrogen ions (Protons, H+) from the inside to the outside of the inner mitochondrial membrane. Each of the respiratory chain complexes I, III and IV acts as a proton pump. The inner membrane is impermeable to ions in general but particularly to protons, which accumulate outside the membrane, creating an electrochemical potential difference across the membrane (ΔμH+). This consists of a chemical potential (difference in pH) and an electrical potential. The electrochemical potential resulting from the asymmetric distribution of the hydrogen ion is used to drive the mechanism responsible for the formation of ATP (Fig. 10.4).
Experimental Evidences to Support the Chemiosmotic Hypothesis
• Addition of protons H+ (acid) to the external medium of intact mitochondria leads to the generation of ATP. • Oxidative phosphorylation does not take place in soluble system where there is no possibility of a vectorial ATP synthase.
• A closed membrane is a must to achieve oxidative phosphorylation.
ATP SYNTHASE
Much information is now available regarding ATP synthase and its role in ATP formation.
SECTION TWO
Structure: (Fig. 10.5)
It is an enzyme complex present in the inner mitochondrial membrane. It is now referred as COMPLEX V the enzyme complex has two subunits – F0 and F1.
• F0 Unit or Subcomplex
It spans inner mitochondrial membrane and serves as a proton channel through which protons enter into mitochondria.
It is a disk of C-subunits. Attached to it is a γγγγγ-subunit in the form of a bent axle. The γγγγγ-subunit fits inside the F1 subcomplex.
• F1 Unit or Subcomplex
This projects into the mitochondrial matrix. It catalyses the ATP synthesis. F1 subcomplex consists of 3βββββ chains (βββββ3) and 3ααααα chains (ααααα3).
γ-subunit fits inside the F1 subcomplex of 3α and 3β subunits which are fixed to the membrane.
In addition, the complex has sigma and epsilon subunits, the function of which are not known.
Mechanism of ATP Synthesis (Boyer’s hypothesis)
Paul Boyer originally proposed a binding change mechanism.
According to this hypothesis 3β subunits (catalytic sites) though structurally similar, but functionally not same at a particular time.
It is envisaged that βββββ-subunits occur in 3 forms: • ‘O’ form (Open form): It has low affinity for
substrates ADP + Pi.
• ‘L’ form (Loose form): Can bind substrates ADP and Pi with more affinity but catalytically it is inactive.
• ‘T’ form (Tight form): Binds substrates ADP and Pi tightly and catalyses ATP synthesis.
Protons entering the system cause conformational changes in the βββββ -subunits.
Rotary or Engine Driving Model
Original Boyer’s hypothesis is now modified. It is now widely accepted that protons passing through the disk of ‘C’ subunits of F0 subcomplex cause it and the attached γ-subunit to rotate. The βββββ-subunits which are fixed to membrane do not rotate.
ADP and Pi are taken up sequentially by the β-subunits which undergo conformational changes.
‘O’ form → ‘L’ form → ‘T’ form
↑ ↑
and forms ATP, which is expelled as the rotating γ-subunit squeezes each β-subunit inturn.
Thus 3 ATP molecules are generated per revolution. Inhibitors of Oxidative Phosphorylation
• Oligomycin: It binds with the enzyme ATP synthase and blocks the proton channels. It thus prevents the translocation of H+ into the mitochondrial matrix, this leads
to accumulation of H+ at higher concentration in
intermembrane space. Since protons cannot be pumped out against steep proton gradients, electron transport stops (respiration stops).
• Atractyloside: It is a glycoside, it blocks the translocase that is responsible for movement of ATP and ADP, across the inner mitochondrial membrane. Adequate supply to ADP is blocked thus preventing phosphoglation and ATP formation (Refer Fig. 10.1).
• Bongregate: Toxin produced by Pseudomonads. It acts similarly to atractyloside.