CHEMIOSMOSIS & UNCOUPLERS PROTEINS DR SAMEER FATANI

CHEMIOSMOSIS Is the diffusion of ions across a selectively-permeable membrane. More specifically, it relates to the generation of ATP by the movement of hydrogen ions across a membrane during cellular respiration. Hydrogen ions(protons) will diffuse from an area of high proton concentration to an area of lower proton concentration. It has been proposed that an electrochemical concentration gradient of protons across a membrane could be harnessed to make ATP. This process is referred to osmosis, the diffusion of water across a membrane, which is why it is called chemiosmosis. ATP synthase is the enzyme that makes ATP by chemiosmosis. It allows protons to pass through the membrane using the kinetic energy to phosphorylate ADP making ATP.

The Chemiosmotic Theory The theory suggests essentially that most ATP synthesis in respiring cell come from the electrochemical gradient across the inner membranes of mitochondria by using the energy of NADH and FADH2 formed from the breaking down of energy rich molecules such as glucose. The oxidation of acetyl CoA in the mitochondrial matrix is coupled to the reduction of a carrier molecule such as NAD and FAD. The carriers pass electrons to the electron transport chain(ETC) in the inner mitochondrial membrane, which in turn pass them to other proteins in the ETC. The energy available in the electrons is used to pump protons from the matrix across the inner mitochondrial membrane, storing energy in the form of a transmembrane electrochemical gradient. The protons move back across the inner membrane through the enzyme ATP synthase. The flow of protons back into the matrix of the mitochondrion via ATP synthase is enough energy for ADP to combine with inorganic phosphate to form ATP. The electrons and protons at the last pump in the ETC are taken up by oxygen to form water.

Oxidative phosphorylation ATP synthase then uses the energy stored in this gradient to make ATP. This process is called oxidative phosphorylation because oxygen is the final electron acceptor and the energy released by reducing oxygen to water is used to phosphorylate ADP and generate ATP.

The proton-motive force In all cells, chemiosmosis involves the proton-motive force (PMF) in some step. This can be described as the storing of energy as a combination of a proton and voltage gradient across a membrane. The chemical potential energy refers to the difference in concentration of the protons and the electrical potential energy as a consequence of the charge separation (when the protons move without a counter-ion). In most cases the proton motive force is generated by an electron transport chain which acts as both an electron and proton pump, pumping electrons in opposite directions, creating a separation of charge. In the mitochondria, free energy released from the electron transport chain is used to move protons from the mitochondrial matrix to the intermembrane space of the mitochondrion. Moving the protons to the outer parts of the mitochondrion creates a higher concentration of positively charged particles, (then electrical potential gradient is about-200 mV (inside negative). This charge difference results in an electrochemical gradient. This gradient is composed of both the pH gradient and the electrical gradient. The pH gradient is a result of the H+ ion concentration difference.

(accumulation of hydrogen ions) Mechanism of proton-motive force + - mitochondria energy Energy released from ETC create charge separation (accumulation of hydrogen ions) The same energy create electrical potential gradient

OF OXIDATIVE PHOSPHORYLATION UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and oxidative phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase. Introduction ATP is synthesized in energy-transducing membranes such as those of mitochondria. The gradient energy necessary to produce ATP in the mitochondria is derived from E.T.C., which then (the energy) passed onto another type of reactions called oxidative phosphorylation to produce ATP. Thus, ATP is synthesized by coupling two reactions, electron transport and phosphorylation. Uncouplers inhibit ATP synthesis by preventing this coupling reaction in such a fashion that the energy produced by redox reactions cannot be used for phosphorylation. Thus, in the presence of an uncoupler, the activities of electron flow and ATPsynthase are not inhibited, but ATP synthesis cannot take place.

Oxidative phosphorylation Oxidative phosphorylation Reactions of E.T.C. (electron transport) Oxidative phosphorylation (ATP synthesis) Gradient energy Normal cellular respiration Reactions of E.T.C. (electron transport) Oxidative phosphorylation (NO ATP synthesis) Gradient energy Uncoupling of cellular respiration Uncoupler protein

Features of uncouplers: A wide variety of compounds are known to be uncouplers of oxidative phosphorylation in mitochondria. Most of them are hydrophobic weak acids that act by protonophoric action and activities;i.e, activities for transporting H+ through an H+ -impermeable membrane. And considered as nonsite-specific active compounds acting on biomembranes. ATP is synthesized from ADP and Pi when H+ enters the mitochondria via H+ -ATPsynthase. Thus, the protonophoric action is to collapse and shrinking the H+ chemical potential gradient by transport of H+ into the mitochondria via the membrane, which regarded as essential for uncoupling action.   This suggests that these uncouplers act on membranes and not directly on the ATPsynthase protein.

The mechanism of Protonophoric Activity of Weakly Acidic Uncouplers At the membrane-water interface, the anionic form of the uncoupler, U- , traps H+ and becomes the neutral form UH. UH traverses the membrane to the opposite side where it releases H+. U- then returns to the original interface where it again traps H+. By this uncoupler cycle, H+ is transported into the inner side of mitochondria through the H+ impermeable membrane, thus dissipating the H+ gradient across the membrane, which results in uncoupling.

Protonophoric action of weakly acidic uncoupler Membrane-water interface Inner mitochondrial membrane Inner side of mitochondria U - UH H + Protonophoric action of weakly acidic uncoupler

Weakly Acidic Uncouplers Act As Nonsite-Specific Bioactive Compounds Acting on Biomembranes The weakly acidic protonophoric uncouplers cause uncoupling primarily by interaction of the weakly acidic uncoupler with the phospholipid in the target membrane, making the membrane permeable to H+, which results in uncoupling. The mechanisms of induction of biological activity can be classified into two types: 1- In one case compounds interact with a specific receptor site in the membrane. These bioactive compounds are classified as site-specific and their mechanism is summarized as follows: Specific interaction with receptor→ Specific biological activity 2- In the other case compounds interact in a nonsite-specific manner. The mechanism of these compounds can be depicted as: Nonspecific interaction with membranes →Specific biological response Thus, the weakly acidic uncouplers considered as nonsite-specific type compounds, and can show very high potency and specificity, even though they do not have a specific receptor site in the membrane. as observed with SF6847.

EXAMPLES OF UNCOUPLERS 1- Dinitrophenol 2,4-Dinitrophenol (DNP), C6H4N2O5, is a cellular metabolic poison. It uncouples oxidative phosphorylation by carrying protons across the mitochondrial membrane, leading to a rapid consumption of energy without generation of ATP. In living cells, DNP acts as a proton ionophore, an agent that can shuttle protons (hydrogen ions) across biological membranes. It defeats the proton gradient across mitochondrial membrane, collapsing the proton motive force that the cell uses to produce most of its ATP chemical energy. Instead of producing ATP, the energy of the proton gradient is lost as heat. DNP is often used in biochemistry research to help explore the bioenergetics of chemiosmotic and other membrane transport processes.

EXAMPLE OF UNCOUPLERS 2- Thermogenin- brown adipose tissue Thermogenin also called uncoupling protein 1, or UCP1 is an uncoupling protein found in the mitochondria of brown adipose tissue (BAT). It is used to generate heat by non-shivering thermogenesis. Non-shivering thermogenesis is the primary means of heat generation in hibernating mammals and in human infants. The molecular mechanism of UCP1-mediated uncoupling is reasonably well understood; UCP1 provides an alternative pathway by which protons can reenter the mitochondrial matrix, short-circuiting the 'proton circuit' linking the respiratory chain to the ATP synthase (which generates ATP for the cell) and allowing respiration (and hence heat production) to proceed in the absence of ATP synthesis. UCP1 is restricted to brown fat, where it provides a mechanism for the enormous heat-generating capacity of the tissue.