OXIDATIVE PHOSPHORYLATION AND ITS MECHANISM

Oxidative phosphorylation refers to the formation of ATP by oxidation process in the

matrix of mitochondria during the electron transport chain of aerobic respiration which has

been discussed in the previous sub-chapter (page 130-145).

A number of theories have been put forth to explain the mechanism of oxidative

phosphorylation and ATP synthesis. These theories or concepts elucidate how

electrochemical gradients across the mitochordrial membranes may be used to drive ATP

synthesis. These theories give an idea about the energy transduction in mitochondria,

described below.

A. The Chemical Coupling Theory

This theory was originally proposed by Slater in 1953 and subsequently modified by

Mahler and Cordes in 1971. According to Slater, during the oxidation of a compound of

electron transport chain the energy released is trapped by the formation of a bond (wriggle

bond written as ,a high energy bond) between that component and some other molecule

that is not the part of the chain.

However, Mahler and Cordes development a model for respiratory chain-linked

phosphorylation which is analogous to the reactions of substrate-level phosphorylation as

illustrated by the reaction sequences in (i) glycolysis during conversion of 1,3-

bisphosphoglyceric acid to 3-phosphoglyceric acid catalyzed by the enzyme, phospho

glycerate kinase with the production of ATP from ADP and in (ii) Krebs cycle during

conversion of succinyl CoA to succinic acid catalyzed by the enzyme succinly CoA

synthetase with the generation of ATP/GTP from ADP/GDP. In these two reactions ADP is

phosphorylated to ATP at the substrate level (for details, refer glycolysis and Kerbs cycle).

The chemical coupling theory is explained as per the following reaction schemes (Salter,1953)

AH2 + B +I ------- BH2+A~I (Oxidation-reduction reaction)

A~I+X------A+I~X

I~X+Pi------I+X~Pi

X~Pi+ADP------- X+ADP~P

------X + ATP (ATP formation)

[~: high energy bond; A, B; components of electron transport chain ;I, X : postulated

intermediates ; AH2, BH2 : reduced electron carriers]

The chemical coupling theory was not accepted by the plant physiologists because

phosphorylated intermediates or high-energy intermediates of the respiratory carriers have not

yet been identified. Further it fails to consider the role of membranes in which energy

transduction takes place.

B. The Conformational Coupling Theory

This theory was proposed by Boyer et al. (1973) which is based on the concept that an

enzyme undergoes a conformational change when a product is released. ADP is

phosphorylated by Pi to ATP by the action of the enzyme ATPase which is reverse of the

normal catalytic activity of this enzyme in catalyzing the hydrolysis of ATP into ADP and Pi.

This theory suggests that energy is required for a conformational change in the ATPase

which leads to an alternation in its affinity for its substrates, ADP and iP or product, ATP

leading to substrate binding and release of the product as per the following model.

In this model, the sequence of events starts with a loose binding of ADP and Pi (inorganic

phosphate) on ATPase . Next a conformation change in the enzyme protein of

ATPase occurs (Fig. 4,40B) by energy input resulting in a shift from a loose to a tight binding

active site. ATP is formed from ADP and Pi, but its release cannot occur until a second

conformational change alters the active site of ATPase to again come to a loose binding site,

so that the newly synthesized ATP can be released.

Although conformational coupling theory is quite attractive, but it is very difficult for

setting up experiments to test this theory.

C. The Chemiosmotic Theory

This theory was proposed by Peter Mitchell (1961) who obtained Nobel Prize in

Chemistry in 1976. He emphasized the importance of cell membrane in which energy

transduction takes place. This theory is based on the concept of porocity of membrane where

transport of protons (H* ions) takes place like the process of osmosis and also it is analogous

to electricity i.e. the transmission of electrons. For this reason, it is known as chemi-osmotic

theory. It is the most convincing of all the theories to explain the mechanism of oxidative

phosphorylation. It is also applicable to chloroplastic photophosphorylation.

According to chemiosmotic theory, the electron transport chain is coupled with ATP

synthesizing complex only through the membrane potential. The free energy of electron

transport is conserved by pumping H* from the mitochondrial matrix to the intermembrane

space to create an electro-chemical H gradient across the inner mitochondrial

membrane. The electrochemical potential of this gradient is used to syntheisze ATP from

ADP and Pi.

The important observation of chemiosmotic theory are summarized below.

1. As a closed compartment is essential for the generation of electrical potential

gradient the oxidative phosphorylation requires an intact inner mitochondrial

membrane. Synthesis of ATP coupled to electron transfer does not occur in soluble

preparations or in membrane fragments lacking well-defined inside and outside

compartments.

2. The inner mitochondrial membrane is impermeable to ions like H* and OH, whose

free diffusion would discharge the electrochemical gradient.

3. Through the respiratory chain, electron transport results in the transport of protons

into the inter membrane space, creating a measurable electrochemical gradient

across the inner mitochondrial membrane. The pH of outside (intermembrane space)

is 1.4 units lower than that of the matrix and the membrane potential is 0.14 V. Thus

the outside becomes positive and the matrix side becomes negative. The total proton

motive force generated by this process is around 0.22 V which corresponds to a free

energy of 5.2 kcal per mole of proton.

4. Compounds (ionophores) which increase the permeability of the inner membrane to

protons and thercby dissipate the electrochemical gradient allow electron transport to

continue, but inhibit synthesis of ATP. It indicates that they 'uncouple' electron

transport from oxidative phosphorylation. On the other hand, if acidity can be

imposed outside the inner membrane, it stimulates ATP synthesis without electron

transport.

5. Both the respiratory chain and the ATP synthase are vectorially organized in the

inner membrane of mitochondria.

6. The oxidation (removal of e) and reduction (addition ofe) reactions are vectorially

placed an opposite sides of the inner membrane so that there will be a net transport of

protons across the membrane. There are 3 proton-translocating sites mediated by

(a) FMN (Complex D), (b) ubiquinone (UQ), and (c) cytochrome

and these correspond with 3 coupling sites for ATP synthesis.

a3 (Comple IV

The main feature of this theory is a membrane located reversible ATp

synthase

rylation

membrane is mitochondrial inner membrane in case of oxidative phosphor .

chloroplastic inner membrane in case of photophosphorylation. In hydrolytic mod ang

the ATP sythase catalyzes the hydrolysis of ATP to ADP and Pi, but in intacstion

intact system i

catalyzes the reverse reaction i.e., the dehydration of AlDP and Pi to form ATP and

water

) Hydrolytic mode:

ATP Synthase

ADP + Pi

ATP + HO

(i) Intact mode:

ATP+ PiAPSynthase

ATP + HgO

ATP synthase pumps protons out side during hydrolytic made and protons pass inside

(towards matrix) during synthetic or intact made.

The chemiosmotic hypothesis as proposed by Mitchell also predicted the existence of

membrane transporters or specific exchange diffusion carriers which has been later

proved to be correct. These carriers permit reversible exchange of anions (for example C)

for OH and cations (e.g., K*) for H* and regulate the pH and osmotic differential across the

membrane. These systems allow the movement of essential metabolites without breaking the

membrane potential and it is essential for ATP synthase catalyzed reaction in the direction of

ATP synthesis.

III. PHOTOPHOSPHORYLATION

In the process of photosynthesis, the phosphorylation of ADP to form ATP using the

energy of sunlight is called photophosphorylation. Only two sources of energy are available

to living organisms: sunlight and reduction-oxidation (redox) reactions. All organisms

produce ATP, which is the universal enerEy currency of life. Commonly in photosynthesis

this involves photolysis of water and a continuous unidirectional flow of electrons from water

to NADP* through photosystems.

In photophosphorylation, light energy 1s Used to create a high-energy electron donor and a

lower-energy electron acceptor. ElectrOns then move spontaneously from donor to.

through an electron transport cham. In Stroma ol Cnloroplast, ATP is synthesized by the

action of the enzyme ATP synthase, form ADP and iP.

ADP + iP Choroplastic ATP synthase ATP+ H,0

Light

ATP synthase is powered by a transmembrane

ectrochemical potential gradient.

usually in the form of a proton gradient. The functin or the electron transport chain is

produce this gradient. In all living organisms, a series of redox reactions is used to produce a

transmembrane electrochemical potential gradient, or a so called proton motive force (pm).

Redox reactions are chemical reactions in which electrons are transferred from a donor

molecule to an acceptor molecule. Any reaction that decrcases the overall Gibbs free cnergy

of a system will procecd spontaneously, although the reaction may procecd slowly il it is

kinetically inhibited.

The transfer of electrons from a high-energy molecule (the donor) to a low-energy

molecule (the acceptor) can be spatially separated into a series of intermediate redox

reactions. This is an electron transport chain (ETC).

Electron transport chains produce enerEy in the form of a transmembrane clectro-

chemical potential gradient. The gradient can be used to Iransport molecules across

membranes. It can be used to do mechanical work, such as rotating bacterial flagella. It can

be used to produce ATP and NADPH, high-encrgy molecules that are necessary for growth.

Photophosphorylation is of two types, .e., cyclie and non-cyclic photophosphorylation.

which have been described in previous chapter. However, sone additional informations have

been presented below.

(1) Cyclic Photophosphorylation

This form of photophosphorylation occurs in the thylakoid membrane, In cyelic

electron flow, the electron begins from photosy'sterm 1. passes from the primary acceptor to

plheophytin, then to cytochrome bgf (a similar complex to tha lound in nmitochondria), and

then to plastocyanin before returning to chlorophyil. This lransporl chain produees a proton-

motive force, pumping H ions across the membrane, this produces a concenlratiom gradicnt

that can be used to power ATP synthase during chemiosmosis. This pathway is known as

cyclic photophosphorylation, and it produces neither Oz nor NADPH. Unlike non-cyclic

photophosphorylation, NADP+ does not accept the electrons. They are instead sent back to

cytochrome bsf complex (cyt. bg and cyt. f).

In bacterial photosynthesis, a single photosystem is used, and therefore is involved in

cyclic photophosphorylation. It is favoured in anaerobic conditions and conditions of high

irradiance and CO2 compensation points.

2. Non-cyclic Photophosphorylation

The non-cyclic photophosphorylation is a two-stage process involving two different

chlorophyll photosystems. Being a light reaction, non-cyclic photophosphorylation occurs in

the frets or stroma lamellae. First, a water molecule is broken down into 2H++į Oz +2e

by photolysis (or light-splitting). The two electrons from the water molecule are kept l

photosystem II, while the 2H* and O2 are left out for further use. Then a photon is

absorbed by chlorophyll pigments surrOunding the reaction core centre of the photosystem

The light excites the electrons of each pigment, causing a chain reaction that eventuall

transfers energy to the core of photosystem II, exciting the two electrons that are transfered

to the primary electron acceptor, pheophytin. The deficit of electrons is replenished by

taking electrons from another molecule of water. The electrons transfer from pheophytin to

plastoquinone (PQ) which takes the 2e from pheophytin, and two H* atoms from the

stroma and forms PQH2, which later is broken into PQ, the 2e- is released to cytochrome

b6 f complex (cyt. b6 and cyt. f) and the two Ht ions are released into thylakoid lumen. The

electrons then pass through the Cyt b6 and Cyt. f. Then they are passed to plastocyanin (PC)

providing the energy for hydrogen ions (H*) to be pumped into the thylakoid space. Ths

creates a gradient, making H* ions flow back into the stroma of the chloroplast, providing the

energy for the regeneration of ATP.

The photosystem ll complex replaced its lost electrons from an external source. Hvrever.

the two other electrons are not returned to photosystem II as they would in the analogous

cyclic pathway. Instead, the still-excited electrons are transferred to a photosystem I

complex, which boosts their energy level to a higher level using a second solar photon. The

highly excited electrons are transferred to the acceptor molecule, but this time are passed on

to an enzyme called Ferredoxin-NADP* reductase

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