The formation of ATP is done by a cellular pathway which is known as oxidative phosphorylation. The site for the formation of ATP is mitochondria specifically the inner chamber of the mitochondria. Mitochondria is the semi-autonomous organelle of the eukaryotic cell. Mitochondria is the site of various metabolic pathways of the cell including oxidative phosphorylation, which is used for the formation of ATP. The enzymes are distracted between the mitochondrial matrix and the inner membrane of the membrane and its modification that is cristae. It is important to understand the basic concept of oxidative phosphorylation to develop an understanding of the formation and role of ATP in cell metabolism. Oxidative phosphorylation is the reaction in which oxygen is reduced into water. O2 acts as the final electron acceptor accepting the electrons from NADH and FADH2 from the electron transport acceptor. The net result of the reaction is the formation of ATP. It is very important to note that electron transport chain reaction and the ATP formation is coupled, this coupling is driving in fact is the driving force of the ATP synthesis. This article deals with the development of the basic understanding of what is ATP formation, the chemiosmotic model, generation of proton motive force, coupling of ETC, and ATP synthesis. This article also lists some of the examples of the inhibitors of ETC and ATP formation, that can be concluded as inhibitors of oxidative synthesis.
A Brief Overview of ETC
ETC can be defined as the electron transport chain, this is a series of oxidation and reduction reactions involving five complexes. This can be defined as the series of biological molecules that are arranged in a particular fashion to transport electron from various pathways of the cell and transport it to the final electron acceptor that is O2. This transfer of electrons to oxygen is associated with the transfer of the protons from the mitochondrial matrix to the intermembrane space of the mitochondria. The need for pumping protons from matrix to intermembrane space is because to provide the driving force of ATP synthase, the enzyme responsible for the formation of ATP. The driving force of the ATP synthase is called as the proton motive force.
There are the following five complexes associated with the electron transport chain, they are named as, complex I, complex II, complex III, complex IV, and complex V. These complexes are also located in the inner mitochondrial membrane.
Complex I- This complex is also called NADH: oxidoreductase or NADH dehydrogenase. This complex transports electrons from NADH + H+ to the ubiquinol (Q) of complex III. during this, the complex transports four protons from the matrix to the intermembrane space contributing to the proton motive force PMF.
NADH + 5H+ + Q——–> NAD+ +QH2 + 4H+
Complex II- This complex is also known as the succinate dehydrogenase, it transfers electrons from the succinate to ubiquinone of complex III. This complex does not transport electrons to the intermembrane space.
Complex III- this is the complex that receives electrons from both the electron. This is also known as the ubiquinone: cytochrome c oxidoreductase. This complex transfer follows Q- cycle. It transports four protons from the intermembrane space.
QH2 + 2cyt c1(oxidized) + 2H+ ———-> Q + 2cyt c1 (reduced) + 4H+
Complex IV- This is the last complex of the ETC. this is also known as cytochrome oxidase, this complex transfers electrons from cytochrome to the final electron acceptor O2. It transports two protons from the IMS to the matrix.
4cyt c(reduced) + 8H+ + O2——–> 4 cyt c(oxidized) + 2H2O + 4H+
Complex V- It is the complex that is involved in the formation of ATP. ATP synthase is considered as the fifth complex.
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Need for PMF
Proton motive force commonly known as the PMF is generated by the pumping of the proton from the matrix to the intermembrane space. Proton motive force has two components they are, chemical potential energy and electrical potential energy. The chemical potential energy is the result of the concentration gradient created by the protons pumped by the complexes of the electron transport chain. This proton gradient allows the passage of a proton through the ATP synthase enzyme. The electrical gradient is also the result of the protons, the intermembrane space is positively charged whereas the matrix is positively charged. Proton motive force rotates the enzyme which leads to ATP formation. In other words, it is the PMF that drives the rotational catalysis of the enzyme.
It is important to note that ATP synthesis can be performed without the oxidation of the substrate (ETC) if there is in vivo creation of the PMF. thus it is often considered as the coupled reaction. To emphasize the importance of the proton motive force in the formation and role of ATP a mathematical expression is used,
ADP + Pi + nHp ——-> ATP + H2O + nHn
The Net Contribution of Complex to Generate PMF
Complex I- 4 protons
Complex III- 4 protons
Complex IV- 2 protons
Chemiosmotic Model
The chemiosmotic model was first proposed by Peter Mitchell. This model describes the paradigm of the ATP synthesis mechanism. According to this model the electrochemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane, PMF, drives the synthesis of the ATP as the protons flow passively back into the matrix through the proton pore associated with ATP synthase.
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ATP Synthase
ATP synthase is the enzyme that catalyzes the formation of ATP. ATP synthase is an enzyme that has two functional domains, these domains are termed F0 and F1. the enzyme is an F- type ATPase, among the two domains the F0 is the integral domain it is embedded in the inner mitochondrial membrane. The 0 in the F0 depicts the oligomycin sensitive nature of the enzyme. This domain of the enzyme contains a proton pore that allows the proton to move passively from the intermembrane space to the matrix, resulting in the rotational catalysis of the enzyme.
The F1 functional domain is t
he site of reversible binding of ATP and ADP +Pi. It is important to note that the binding is reversible in nature to allow multiple substrates to bind to the enzyme. Stabilization of the binding of ATP compared to ADP +Pi is achieved by the relatively stronger association of the ATP on the enzyme surface. This strong association leads to the release of enough energy to counterbalance the cost of ATP formation in mitochondria.
Unlike all the enzyme-catalyzed reaction the strong association of product to the enzyme leads to the creation of a major energy barrier in the formation and release of the product. The major energy barrier for other enzymes is reaching the transition state whereas in this case, the release of product formed that is ATP is the major energy barrier of the reaction.
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Structure of the ATP Synthase
As described earlier there are two domains of the enzymes, the F1 domain has 9 subunits these subunits can be written as α3 β3γẟε. This denotes that there are three copies of alpha and beta and the rest has only one copy. The beta is the catalytic site where the ATP is bound, gamma is the shaft that is attached to one of the beta subunits of the enzyme. The alpha and beta are arranged in an alternative fashion to produce a knob-like structure.
Fo domain is the oligomycin sensitive domain that has a pore to allow the protons to pass, there are three subunits in this domain, ab2c10-12 the b subunit binds to the ẟ subunit of the F1 domain. The c subunit is hydrophobic and contains 2 transmembrane alpha-helix, these form the disc-like structure of the enzyme.
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Rotational Catalysis
Rotational catalysis is the process by which the ATP and ADP +Pi binds to the enzyme and produce ATP. there are three beta copies each copy undertake one of the following conformations that are, beta empty, beta loose, and beta tight. In the empty or open conformation, there is no binding neither of ADP nor ATP. in the loose conformation the enzyme is bound to the ADP +Pi. And in the tight, they are bound to the ATP, with every proton that enters through the pore in the (a) subunit of Fo, the gamma shafts rote 120 degrees, leading to the change in the catalytic site and release of the ATP. there are total three 120 degrees movement is required ATP from all three beta subunit.
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Inhibitors
Since the formation of ATP is coupled with the electron transfer chain the inhibitors to the oxidative phosphorylation can inhibit the ATP formation, the table given below list some of the examples of the inhibitors
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Inhibition of Electron Transfer
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Cyanide
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Carbon monoxide
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Antimycin A
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Myxothiazole
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Rotenone
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Pericidin A
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Inhibition of ATP synthase
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Aurovetrin- It inhibits the F1 domain of the ATP synthase
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Oligomycin – It inhibits the Fo domain of the enzyme
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Ventriuricidin- It inhibits the Fo domain of the chloroplast ATP synthase
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DCCD- It blocks the proton flow through the form of mitochondrial and chloroplast ATP synthase.
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Uncouplers of Oxidative Phosphorylation
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FCCP
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DNP
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Valinomycin
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Thermogenin