5. Bioenergetics

Life Sciences - LS (Molecules and their interaction relevant to Biology) 5. BIOENERGETICS

Oxidative Phosphorylation Oxidative phosphorylation (or OXPHOS in short) is the metabolic pathway in which the mitochondria in cells use their structure, enzymes, and energy released by the oxidation of nutrients to reform ATP. Although the many forms of life on earth use a range of different nutrients, ATP is the molecule that supplies energy to metabolism. Almost all aerobic organisms carry out oxidative phosphorylation. Oxidative phosphorylation began with the report in 1906 by Arthur Harden of a vital role for phosphate in cellular fermentation, but initially only sugar phosphates were known to be involved. 1940s, Herman Kalckar work on this between the oxidation of sugars and the generation of ATP was firmly established, confirming the central role of ATP in energy transfer that had been proposed by Fritz Albert Lipmann in 1941. Later, in 1949, Morris Friedkin and Albert L. Lehninger proved that the coenzyme NADH linked metabolic pathways such as the citric acid cycle and the synthesis of ATP. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis. 5.1 Electron Transport Chain The electron transport chain (aka ETC) is a process in which the NADH and [FADH ] produced  2 during glycolysis, -oxidation, and other catabolic processes are oxidized thus releasing energy in the form of ATP. The mechanism by which ATP is formed in the ETC is called chemiosmotic phosphorylation.

Fig. : Electron Transport Chain Overview The electron transport chain is present in multiple copies in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. In the diagram located above there are the major electron transporters responsible for making energy in the ETC. Contact Us : Website : www.eduncle.com | Email : [email protected] | Call Toll Free : 1800-1201021 176 Life Sciences - LS (Molecules and their interaction relevant to Biology)

NADH-coenzyme ubiquinone oxidoreductase (complex I)  An integral protein that receives electrons in the form of hydride ions from NADH and passes them on to ubiquinone. The reaction that is catalyzed by this enzyme is the two electron oxidation of NADH by coenzyme Q10 or ubiquinone (represented as Q in the equation below), a lipid-soluble quinone that is found in the mitochondrion membrane:  +  + + NADH + Q + 5 H matrix NAD + QH2 + 4H intermembrane  The start of the reaction, and indeed of the entire electron chain, is the binding of a NADH molecule to complex I and the donation of two electrons. The electrons enter complex I via a prosthetic group attached to the complex, flavin mononucleotide (FMN). The addition

of eletrons to FMN converts it to its reduced form, FMNH2. The electrons are then transferred through a series of iron-sulfur clusters: the second kind of prosthetic group present in the complex. There are both [2Fe-2S] and [4Fe-4S] iron-sulfur clusters in complex I.  As the electrons pass through this complex, four protons are pumped from the matrix into the intermembrane space. Finally, the electrons are transferred from the chain of iron- sulfur clusters to a ubiquinone molecule in the membrane. Reduction of ubiquinone also contributes to the generation of a proton gradient, as two protons are taken up from the

matrix as it is reduced to ubiquinol (QH2). Succinate-Q oxidoreductase (complex II)  (Succinate-ubiquinone oxidioreductase aka succinate dehydrogenase from the TCA cycle): A peripheral protein that receives electrons from succinate (an intermediate metabolite of

the TCA cycle) to yield fumarate and [FADH2]. From succinate the electrons are received

by [FAD] (a prosthetic group of the protein) which then become [FADH2]. The electrons are then passed off to ubiquinone.  Also known as complex II or succinate dehydrogenase, is a second entry point to the electron transport chain. It is unusual because it is the only enzyme that is part of both the citric acid cycle and the electron transport chain.  It oxidizes succinate to fumarate and reduces ubiquinone. As this reaction releases less energy than the oxidation of NADH, complex II does not transport protons across the membrane and does not contribute to the proton gradient.  Succinate + Q Fumarate + QH2 In some eukaryotes, such as the parasitic worm Ascaris suum, an enzyme similar to complex II, fumarate reductase (menaquinol:fumarate oxidoreductase, or QFR), operates in reverse to oxidize ubiquinol and reduce fumarate. Q (Ubiquinone/ubiquinol) : Ubiquinone (the oxidized form of the molecule) receives electrons from several different carriers; from I, II, Glycerol-3-phosphate dehydrogenase, and ETF. It is now the reduced form (ubiquinol) which passes its electron off to III. Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Q oxidoreductase), also known as electron transferring-flavoprotein dehydrogenase, is a third entry point to the electron transport chain. It is an enzyme that accepts electrons from electron-transferring flavoprotein in the mitochondrial matrix, and uses these electrons to reduce ubiquinone. This enzyme contains a flavin and a [4Fe-4S] cluster, but, unlike the other respiratory complexes, it attaches to the surface of the membrane and does not cross the lipid bilayer.  ETFred + Q ETFox + QH2  In mammals, this metabolic pathway is important in beta oxidation of fatty acids and catabolism of amino acids and choline, as it accepts electrons from multiple acetyl- CoAdehydrogenases. In plants, ETF-Q oxidoreductase is also important in the metabolic responses that allow survival in extended periods of darkness.

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Q-cytochrome c oxidoreductase (complex III) : An integral protein that receives electrons from ubiquinol which are then passed on to Cytochrome c.  The reaction catalyzed by complex III is the oxidation of one molecule of ubiquinol and the reduction of two molecules of cytochrome c, a heme protein loosely associated with the mitochondrion. Unlike coenzyme Q, which carries two electrons, cytochrome c carries only one electron.  +  + QH2 + 2 Cyt cax + 2 H matrix Q + 2 Cyt cred + 4 H intermembrance Cytochrome c oxidase (complex IV) : An integral protein that receives electrons from Cytochrome c and transfers them to oxygen to produce water within the mitochondria matrix.  Cytochrome c oxidase, also known as complex IV, is the final protein complex in the electron transport chain. The mammalian enzyme has an extremely complicated structure and contains 13 subunits, two heme groups, as well as multiple metal ion cofactors - in all, three atoms of copper, one of magnesium and one of zinc.  This enzyme mediates the final reaction in the electron transport chain and transfers electrons to oxygen, while pumping protons across the membrane. The final electron acceptor oxygen, which is also called the terminal electron acceptor, is reduced to water in this step. Both the direct pumping of protons and the consumption of matrix protons in the reduction of oxygen contribute to the proton gradient. The reaction catalyzed is the oxidation of cytochrome c and the reduction of oxygen:  +  + 4 Cyt Cred + O2 + 8 H matrix 4Cyt cox + 2H2O + 4H intermembrance ATP Synthase (complex V) : An integral protein consisting of several different subunits. This protein is directly responsible for the production of ATP via chemiosmotic phosphorylation. It uses the proton gradient created by several of the other carriers in the ETC to drive a mechanical rotor. The energy from that rotor is then used to phosphorylate ADT to ATP.  +    ADP+ Pi+ 4 H intermembrance ATP H2O 4Hmatrix  The portion embedded within the membrane is called FO and contains a ring of c subunits and the proton channel. The stalk and the ball-shaped headpiece is called F1 and is the site of ATP synthesis.  The ball-shaped complex at the end of the F1 portion contains six proteins of two different   kinds (three subunits and three subunits), whereas the "stalk" consists of one protein:    the sub unit, with the tip of the stalk extending into the ball of and subunits.     Both the and subunits bind nucleotides, but only the subunits catalyze the ATP synthesis reaction. Reaching along the side of the F1 portion and back into the membrane   is a long rod-like subunit that anchors the and subunits into the base of the enzyme.

Fig. : ATP Synthesis Reaction Contact Us : Website : www.eduncle.com | Email : [email protected] | Call Toll Free : 1800-1201021 178 Life Sciences - LS (Molecules and their interaction relevant to Biology)

This ATP synthesis reaction is called the binding change mechanism and involves the active site  of a subunit cycling between three states. In the "open" state, ADP and phosphate enter the active site. The protein then closes up around the molecules and binds them loosely. The enzyme then changes shape again and forces these molecules together, with the active site in the resulting "tight" state. binding the newly produced ATP molecule with very high affinity. Finally, the active site cycles back to the open state, releasing ATP and binding more ADP and phosphate, ready for the next cycle. In some bacteria and archaea, ATP synthesis is driven by the movement of sodium ions through the cell membrane, rather than the movement of protons. Archaea such as Methanococcus also contain the A1Ao synthase, a form of the enzyme that contains additional proteins with little similarity in sequence to other bacterial and eukaryotic ATP synthase subunits. 5.2 Alternative Reductases and Oxidases Many eukaryotic organisms have electron transport chains that differ from the much-studied mammalian enzymes described above. For example, plants have alternative NADH oxidases, which oxidize NADH in the cytosol rather than in the mitochondrial matrix, and pass these electrons to the ubiquinone pool. These enzymes do not transport protons, and, therefore, reduce ubiquinone without altering the electrochemical gradient across the inner membrane. Another example of a divergent electron transport chain is the alternative oxidase, which is found in plants, as well as some fungi, protists, and possibly some animals. This enzyme transfers electrons directly from ubiquinol to oxygen. The advantages produced by a shortened pathway are not entirely clear. However, the alternative oxidase is produced in response to stresses such as cold, reactive oxygen species, and infection by pathogens, as well as other factors that inhibit the full electron transport chain. 5.3 Prokaryotic electron transport chains The main difference between eukaryotic and prokaryotic oxidative phosphorylation is that bacteria and archaea use many different substances to donate or accept electrons. This allows prokaryotes to grow under a wide variety of environmental conditions. In E. coli, for example, oxidative phosphorylation can be driven by a large number of pairs of reducing agents and oxidizing agents, which are listed below. The midpoint potential of a chemical measures how much energy is released when it is oxidized or reduced, with reducing agents having negative potentials and oxidizing agents positive potentials. 5.4 Inhibitors Several well-known drugs and toxins that inhibit oxidative phosphorylation. Although any one of these toxins inhibits only one enzyme in the electron transport chain, inhibition of any step in this process will halt the rest of the process. For example, if oligomycin inhibits ATP synthase, protons cannot pass back into the mitochondrion. As a result, the proton pumps are unable to operate, as the gradient becomes too strong for them to overcome. NADH is then no longer oxidized and the citric acid cycle ceases to operate because the concentration of NAD+ falls below the concentration that these enzymes can use.

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Compounds Use Site of action Effect on oxidative phosphorylation Cyanide Poisons Complex IV Inhibit the electron transport chain by binding more monoxide Fe–Cu center in Carbon strongly than oxygen to the Azide cytochrome c oxidase, preventing the reduction of Hydrogen sulfide oxygen. Oligomycin Antibiotic Complex V Inhibits ATP synthase by blocking the flow of protons subunit. through the Fo that disrupt the proton gradient by CCCP Poisons, Inner Ionophores [2,4-Dinitrophenol] weight- membrane carrying protons across a membrane. This uncouples proton pumping from ATP loss[N1] ionophore synthesis because it carries protons across the inner mitochondrial membrane. Rotenone Pesticide Complex I Prevents the transfer of electrons from complex I to ubiquinone by blocking the ubiquinone-binding site. andoxalo Malonate Poisons Complex II Competitive inhibitors of succinate dehydrogenase acetate (complex II). cytochrome c reductase, Antimycin A Piscicide Complex III Binds to the Qi site of oxidation of ubiquinol. thereby inhibiting the Coupled Reaction Energy coupling in mechanical and chemical processes. 1. The downward motion of an object releases potential energy that can do mechanical work. The potential energy made available by spontaneous downward motion, an exergonic process (pink), can be coupled to the endergonic upward movement of another object (blue). 2. In reaction 1, the formation of glucose-6-phosphate from glucose and inorganic phosphate (Pi) yields a product of higher energy than the two reactants. For this endergonic reaction, DG is positive. In reaction 2, the exergonic breakdown of adenosine triphosphate (ATP) can drive an endergonic reaction when the two reactions are coupled.  The exergonic reaction has a large, negative free-energy change ( G2), and the endergonic reaction has  a smaller, positive change ( G1). The third reaction accomplishes the sum of reactions 1 and 2, and    the free-energy change, AGs, is the arithmetic sum of G1 and G2. Because G3 is negative, the overall reaction is exergonic and proceeds spontaneously. Reaction 1 : Condensation of glucose (alcohol) with inorganic phosphate ion (acid) to make glucose-6-phosphate (an ester)

Glucose + Pi <=> glucose-6-phosphate + H2O  ’ = + 13.8 kJ/mol, endergonic) ( Go Reaction 2 : Hydrolysis of ATP, a phosphoanhydride, to generate ADP and inorganic phosphate  o’ = -30.5 kJ/mol, exergonic). ATP + H2O <=> ADP + Pi ( G To couple the 2 reactions (which requires some chemical mechanism, of course), add reactants on left,  ‘ values to get  ‘ for coupled reaction: add products on right, and add Go Go Glucose + ATP < = > glucose-6-phosphate + ADP  º' = –16.7 kJ/mol) ( G   G < 0 G < 0

Work Loss of done potential raising energy of object position

Endergonic Exergonic Fig. : Mechanical Process Contact Us : Website : www.eduncle.com | Email : [email protected] | Call Toll Free : 1800-1201021 180 Life Sciences - LS (Molecules and their interaction relevant to Biology)

Reaction 2  Reaction 3 ATP ADP + Pi  Glucose + ATP

G Glucose-6-Phosphate + ADP 

, Reaction 1 y

g 

r Glucose + Pi e

n Glucose-6-Phosphate

E  G3

e  G e 2 r

F  G1    G3 = G1 + G2

Reaction Coordinate Fig. : Chemical Process  º' (kJ × mol–1) (a) G Endergonic half-reaction 1 Pi + Glucose Glucose-6-Phosphate + H2O +13.8 Exergonic half-reaction 2 –30.5 ATP + H2O ADP + Pi Overall coupled reaction ATP + Glucose ADP + Glucose-6-phospate –16.7  º' (kJ × mol–1) (b) G Exergonic – COO O – CH2 C + H2O CH3 C COO + Pi –61.9 half-reaction 1 – 2 OPO3 Pyruvate Phosphoenol Pyruvate Endergonic half-reaction 2 ADP + Pi ATP + H2O +30.5

– COO O Overall – CH2 C + ADP –31.4 CH3 C COO + ATP – Coupled Reaction 2 OPO3 Group Transfer The transfer of acyl, glycosyl and phosphoryl groups from one nucleophile to another is common in living cells. Acyl group transfer generally involves the addition of a nucleophile to the carbonyl carbon of an acyl group to form a tetrahedral (to move) intermediate.

– O O O

C R C X C R X R X Y – :Y :X Fig. : Tetrahedral Intermediate Phosphoryl group transfers play a special role in metabolic pathways. A general theme in metabolism ‘activate’ the intermediate for is the attachment of a good leaving group to a metabolic intermediate to subsequent reaction. Among the better leaving groups in nucleophilic substitution reactions are inorganic –  4 2 orthophosphate (the ionized form of H3PO4 at neutral pH, a mixture ofH2PO and HPO 4 ) and inorganic  4 pyrophosphate (P2O 7 ); esters and anhydrides of phosphoric acid are effectively activated for reaction. Contact Us : Website : www.eduncle.com | Email : [email protected] | Call Toll Free : 1800-1201021 181 Life Sciences - LS (Molecules and their interaction relevant to Biology)

Nucleophilic substitution is made more favourable by the attachment of a phosphoryl group to an – otherwise poor leaving group such as OOH. Nucleophilic substitutions in which the phosphoryl group (  2 PO 3 ) serves as a leaving group occur in hundreds of metabolic reactions. The large families of enzymes that catalyze phosphoryl group transfers with ATP as donor are ‘to move), hexokinase, ‘moves’—a phosphoryl group from ATP to glucose. called kinases (Greek kinein Biological Energy Transducers Cells contain a variety of molecular transducers, which convert the energy of electron flow into useful ‘circuit,’ with a relatively reduced compound such as work. Living cells have an analogous biological glucose as the source of electrons. As glucose is enzymatically oxidized, the released electrons flow spontaneously through a series of electron-carrier intermediates to another chemical species, such as O2.

This electron flow is exergonic, because O2 has a higher affinity for electrons than do the electron-carrier intermediates. The resulting electromotive force provides to a variety of molecular energy transducers (enzymes and other proteins) that do biological work. In the mitochondrion, for example, membrane-bound enzymes couple electron flow to the production of a transmembrane pH difference, accomplishing osmotic and electrical work. The proton gradient thus formed has potential energy sometimes called the proton- motive force by analogy with electromotive force. Another enzyme, ATP synthase in the inner mitochondrial membrane, uses the proton motive force to do chemical work: synthesis of ATP from ADP and Pi as protons flow spontaneously across the membrane. Similarly, membrane-localized enzymes in E.coli convert electromotive force to proton-motive force, which is then used to power flagella motion. The principles of electrochemistry that govern energy changes in the macroscopic circuit with a motor and battery apply with equal validity to the molecular processes accompanying electron flow in living cells. NADH and NADPH It act with dehydrogenases as soluble electron carriers. Nicotinamide adenine dinucleotide (NAD+) in its oxidized form) and its close analog nicotinamide adenine dinucleotide phosphate (NADP+) are composed of two nucleotides joined through their phosphate groups by a phosphoanhydride bond. Because the nicotinamide ring resembles pyridine, these compounds are sometimes called pyridine nucleotides. The vitamin niacin is the source of the nicotinamide moiety in nicotinamide nucleotides. Both coenzymes undergo reversible reduction of the nicotinamide ring. O O O H – H H H H C 2e C C NH + NH2 NH2 or 2 + H + .. .. N 2H+ N N O- CH2 O R A side R B side – H H NADH O P O H H (reduced) OH OH O NH 2 1.0 Oxidized – + N e (NAD ) O P O N Adenine c 0.8 n a b CH O N N r 0.6 O 2 o s Reduced b

H H A 0.4 (NADH) H H NAD+ 0.2 (oxidized) OH OH 0.0 In NADP+ this hydroxyl group 220 240 260 280 300 320 340 360 380 is esterified with phosphate Wavelength (nm) (a) (b) Fig. : NAD and NADP : (a) Nicotinamide adenine dinucleotide, NAD+ and its phosphorylated analong NADP+ undergo reduction to NADH and NADPH, accepting a hydride ion. (b) The UV absorption spectra of NAD+ and NADH. Reduction of the nicotinamide ring produces a new, broad absorption band with a maximum at 340 nm. Contact Us : Website : www.eduncle.com | Email : [email protected] | Call Toll Free : 1800-1201021 182 Life Sciences - LS (Molecules and their interaction relevant to Biology)

More than 200 enzymes are known to catalyze reactions in which NAD+ (or NADP+ ) accepts a hydride ion from a reduced substrate NADPH (or NADH) donates a hydride ion to an oxidized substrate. Flavin Nucleotides Bound in Flavoproteins Flavoproteins are enzymes that catalyze oxidation- reduction reactions using either flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as coenzyme. These coenzymes, the flavin nucleotides, are derived from the vitamin riboflavin. The flavin nucleotide in most flavoproteins is bound rather tightly to the protein and in some enzymes, such as succinate dehydrogenase, it is bound covalently. Such tightly bound coenzymes are properly called prosthetic groups. They do not transfer electrons by diffusing from one enzyme to another; rather, they provide a means by which the flavoproteins can temporarily hold electrons while it catalyzes electron transfer from a reduced substrate to an electron acceptor. One important feature of the flavoproteins is the variability in the standard reduction potential (Eo) of the bound flavin nucleotide. Certain flavoproteins act in a quite different role as light receptors. Cryptochromes are a family of flavoproteins, widely distributed in the eukaryotic phyla that mediate the effects of blue light on plant development and the effects of light on mammalian circadian rhythms (oscillations in physiology and biochemistry, with a 24-hour period). The cryptochromes are homologs of another family of flavoproteins, the photolyases.

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