Phosphorylation oxidative

OXIDATIVE PHOSPHORYLATION The Concept of High Energy Phosphate 

The unification and direction which the P concept gave to metabolic studies led to its rapid and enthusiastic adoption by the new generation of biochemists starting work after the end of World War II. Kalckar suggested the contribution resonance stabilization might make to the distinction between the two classes of phosphate compounds. T.L. Hill and Morales (1951) identified ionization effects and electrostatic repulsion as further possible contributants to the net differences in free energy between products and reactants. 

Both Kalckar and the Russians measured the ratio of atoms of phosphorus yielding ATP to the atoms of oxygen utilized (P/O ratio) and found values significantly greater than 1, the figure expected if a phosphate group had been introduced into a cycle intermediate, as in the glycolytic pathway. 

Interpretation of these early experiments with crude tissue preparations was greatly complicated by the presence of very active phosphatases (ATPases) which rapidly hydrolyzed any ATP which might have been formed. Ochoa suggested that the amount of Pj apparently esterified should be corrected for the measurable rate of ATP hydrolysis by the preparation. This gave P/O ratios approaching 3. Belitzer and Tsibakowa and Ochoa realized that phosphorylation must occur not only when the substrate is dehydrogenated. .. but also during 

With these improved techniques P-hydroxybutyrate, which penetrates mitochondria easily and is oxidized to acetoacetate using NAD+ as H acceptor, gave a P/O ratio of 3, the value equivalent to that from the reoxidation of NADH found by Lehninger. Succinate, which bypassed the NAD+/NADH step, gave a ratio of 2. When cytochrome c-Fe2+ was 

3 Oxidative Phosphorylation. Oxidative phosphorylation, that is the production of ATP during the passage of electrons down the terminal electron transport chain, may be disrupted in two distinct ways. Compounds that divorce the process of electron transport and the phosphorylation of ADP are termed uncoupling agents. They permit NADH and succinate to be oxidised via the electron transport chain without the production of ATP and are lethal. Oxidative phosphorylation may also be inhibited directly, thus preventing the oxidation of NADH and succinate. Several products are available that exploit these modes of action. Characteristically, they have wide activity spectra that span major disciplines of pesticide use. 

The triphenyltins fentin acetate and fentin hydroxide are non-systemic 

Fentin hydroxide has been demonstrated to inhibit oxidative phosphorylation in rat liver mitochondria and it is thought that the membrane-located component of ATPase is the target for triphenyltin fungicides. 

1 Evidence that oxidative reactions can drive the for-— ji mation of ATP was obtained about 1940 by Herman 

Approximately 2.5 molecules of ADP can be phosphorylated to ATP for each pair of electrons that traverse the electron-transport chain from NADH to 02. About 1.5 molecules of ATP are formed for a pair of electrons that enter the chain via succinate dehydrogenase or other flavoproteins such as glycerol-3-phosphate dehydrogenase. Approximately one molecule of ATP is formed for each pair of electrons that enters via cytochrome c. Electron flow through each of complexes I, III, and IV thus is coupled to phosphorylation. 

How do mitochondria couple such different types of reactions as electron transfer and the formation of a phosphate anhydride bond We explore this question in the following sections. 

The chemi-osmotic theory of oxidative phosphorylation has been reviewed,74 a model for mitochondrial oxidative phosphorylation in which a membrane potential or proton gradient might transmit energy from an oxidation step to ATP synthesis has been proposed,76 and adenine nucleotide transport in mitochondria has been reviewed.76 

In a chemical model for oxidative phosphorylation77 the anaerobic oxidation of iV-benzyl 1,4-dihydronicotinamide by a pyridine solution of haemin was accompanied by the synthesis of ATP from ADP and inorganic phosphate. In support of an alternative chemical model involving sulphenyl phosphates as the reactive species,78 lipophilic thioureas have been shown to inhibit mitochondrial oxidative phos- 

The electron transfer processes that occur within the membrane, such as for example, phosphorylation (Fig. 17.6), are well known, but their mechanisms remain unexplained. These electron transfer processes are of primary importance in two types of membrane chloroplasts in photosynthesis, and mitochondria in respiration. 

As an example we use mitochondria (Fig. 17.6). These are small corpuscles that exist in large quantities within cells. They possess an exterior and an interior membrane where the enzymes cytochrome by c, Ci, a and a3, ATPase, and NADH are located. The interior membrane, of non-repetitive structure, contains 80 per cent protein and 20 per cent lipid. The Gibbs free energy variation of the conjugated redox pairs is given by the formal potential, according to 

In the respiratory chain we start from the system NAD/NADH2 (E° = -0.32 V) and reach the system 02/H20 ( 0 = +0.82 V). The free energy change is thus —220 kJ mol-1, but this tells us nothing about the mechanism of action of the mitochondria. 

Some of the steps of the electron transfer mechanism in biological membranes are known, as they are for the associated proton transfer 

In a chemical model for mitrochondrial oxidative phosphorylation/ it has been proposed that the mitochondrial membrane, to which ATP and inorganic phosphate are attached, is held in an extended inactive form (50) by coulombic repulsion of positive charges. On reduction of the membrane by NADH one positive centre is removed, and folding of the membrane can occur with extrusion of water. This creates a non-aqueous environment around the ADP (51) and a metal ion can now catalyse the formation 

A dynamic model has been proposed for the synthesis of ATP during oxidative phosphorylation in which ADP and inorganic phosphate combine directly to give a quinquecovalent intermediate. While such a model may be valuable in drawing attention to the possible participation of quinquecovalent intermediates in ATP synthesis, no mention is made of the role of oxidation in this reaction. 

The synthesis of ADP and ATP by the aerial oxidation of ferro-haemo-chrome solutions is knownand the participation of an imidazole 

Hydroquinone phosphates (57) transfer phosphate to substrates following 

As mentioned in Chapter 8, a mechanism for ATP synthesis involving a quinquecovalent intermediate has been put forward in a review which emphasizes that the phosphorylation mechanism is mechanistically indepen- 

Roisch and F. Lingens, Angew. Chem. Internat. Edit., 1974,13, 400. 

The major role of electron transfer is the generation of ATP from ADP and P, (oxidative phosphorylation). Since the 

CH 9 OXIDATION OF FUELS AND ATP GENERATION PHYSIOLOGICAL AND CLINICAL IMPORTANCE 

A second, related, question is, what is the mechanism of this coupling The answer, which is central to the process of oxidative phosphorylation, is now presented. 

Coupling of electron transfer with oxidative phosphorylation 

A major conceptual advance was made when it was appreciated that the coupling between electron transfer and 

From the discussions above it follows that up to the 1980s sufficient evidence had accumulated, indicating a potential equipment of propioni-bacteria for aerobic life. One of the essential steps was the discovery of oxidative phosphorylation in propionic acid bacteria (Bruchatcheva et al., 1975). 

Oxidative phosphorylation was also demonstrated by an indirect method, by estimating how much substrate was used for the synthesis of one unit of biomass (Ts) As shown in Table 3.10, the two species display maximal growth yield coefficients (Ys) under aerobic conditions of growth, therefore, in the presence of oxygen the substrate is utilized more effectively, especially by P. petersonii, which produces 2.5 times more ATP per unit substrate under aerobic conditions. In P. shermanii the difference is smaller, indicating that metabolic changes induced by the transition from anaerobic to aerobic conditions are less profound. 

On the basis of similar calculations (7s and 7atp) it has been assumed that anaerobic electron transport to NOs in P. pentosaceum is also linked with oxidative phosphorylation (van Gent-Ruijters et al., 1976). The authors concluded that one mole of ATP is formed when two electrons are transferred from lactate or glycerol 1-phosphate to nitrate (P/NO3 =1), and two moles of ATP are formed when 2e are transferred from NADH to nitrate (P/NOs = 2). 

although both membrane and soluble fractions of P. shermanii and P. petersonii can oxidize NADH, only the oxidation by the particulate fraction is linked with the accumulation of energy. 

The oxidative phosphorylation by the respiratory chains of P. shermanii and P. petersonii is sensitive to uncouplers and inhibitors 2,4-DNP (110 M) reduced the P/NADH ratio in both cultures by 20-40% gramicidin (1 10 M) by 60% o-phenanthroline (110 M) by 20% in P. shermanii and by 80% in P. petersonii. A marked inhibition of the oxidative phosphorylation by the iron chelator o-phenanthroline in P. petersonii as compared with P. shermanii may indicate an essential role of non-heme iron in the oxidative phosphorylation of the former. Pretreatment of the particulate fraction of propionibacterial cells by UV-light led to a decrease in P/NADH ratio by 

Of all the intracellular organelles, the mitochondrion has been the most extensively studied with respect to the compartmentation of compounds within its boimdaries. In part, this results from the ease of separation of mitochondria from mammalian tissues (most notably the liver), as well as from the key role mitochondria play in a number of metabolic processes. The mitochondrial membrane is capable of transporting metabolites on specific transporters and of segregating metabolites from the cytosol. It is important to note that some metabolites apparently move across the mitochondrial membrane in an unspecific or non-carrier-linked manner. For example, ketone bodies, water, CO2, and oxygen appear to freely diffuse into and out of mitochondria. In the following sections we will discuss specific aspects of the transport mechanisms, followed by a more general discussion of their role in regulating major metabolic pathways. We will start with the most important result of intracellular compartmentation—oxidative phosphorylation—as viewed by the chemiosmotic theory. 

The placement of the components of the respiratory chain in the inner mitochondrial membrane is of considerable importance to the mechanism of the chemiosmotic theory. Mitchell (1967) calls this the coupling membrane H+, citric-acid-cycle anions, amino acids, and cations can be transported across this membrane by specific carriers imbedded in it. The respiratory chain is folded into three loops  

The mitochondrial inner membrane acts as a barrier to the free movement of ATP and ADP [see Klingenberg (1972) for a review]. Since ATP is formed on the inside of the inner mitochondrial membrane, whereas most of the ATP-utilizing reactions are in the cytosol, the mechanism for ATP translocation is of considerable importance. Klingenberg and his associates (1966, 1972) were the first to clearly describe the transport process and to point out the nature of the specific carrier 

Freshly isolated, intact mitochondria contain considerable amounts of adenine nucleotides which are resistant to removal by repeated washings with isotonic sucrose. This indicates that these compounds are in a compartment—presumably within the inner mitochondrial membrane—which is inaccessible to the sucrose solution. When exogenous adenine nucleotide is added to the mitochondria, there is a rapid exchange with endogenous adenine nucleotides with no net increase in the concentration of adenine nucleotides in the mitochondria. ADP exchanges most rapidly, followed by ATP and then by AMP, which is relatively impermeable. It is the inner mitochondrial membrane through which the adenine nucleotides do not permeate and which contains the specific adenine-nucleotide transporting system. The movement of ATP across the inner mitochondrial membrane (and hence out of the mitochondria) depends directly on the translocation of ADP in the presence of adenylate kinase in the outer compartment of the mitochondria. 

This affords a mechanism for balancing the relative concentration of either of the permeant adenine nucleotides. 

Complex IV spans the membrane, with cytochrome a oriented toward the C side copper ions and cytochrome as are oriented toward the M side. 

Nicotinamide nucleotide transhydrogenase, which catalyzes the reaction NADPH + NAD+ NADH + NADP+, spans the membrane, but its catalytic site faces the M side. 

The anisotropic organization of electron carriers across the membrane accounts for the vectorial transport of protons from the inside to the outside of the membrane, which occurs with the passage of electrons. The coupling of this proton gradient to a proton-translocating ATP synthase (also known as ATP synthetase) accounts for the chemiosmotic coupling in oxidative phosphorylation. 

Flow of reducing equivalents in the respiratory chain and their relationship to energy availability to drive ATP synthesis. The largest free energy changes occur between NADH and FMN, between cytochromes b and ci, and between cytochromes (a + 33) and molecular oxygen ATP formation is coupled to these three sites. 

ATP is synthesized from ADP and phosphate during electron transport in the respiratory chain. This type of phosphorylation is distinguished from substrate-level phosphorylation, which occurs as an integral part of specific reactions in glycolysis and the TCA cycle. The free energy available for the synthesis of ATP during electron transfer from NADH to oxygen can be calculated from the difference in the value of the standard potential of the electron donor system and that of the electron acceptor system. The standard potential of the NADH/NAD+ redox component is —0.32 V and that of H2O/5O2 is -1-0.82 V therefore, the standard potential difference between them is 

Oxidation of thiols or disulphides in pyridine solution in the presence of ADP and inorganic phosphate by two equivalents of iodine or bromine leads to the formation of ATP. The initial reaction between the disulphide and positive halogen is presumably followed by the displacement of halide by phosphate ion to give a phosphorylating species (52). ADP would react with (52) with the formation of ATP and a thiosulphinic 

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The term vitamin K2 was applied to 2-methyl-3-difarnesyl-l,4-naphthoquinone, m.p. 54 C, isolated from putrefied fish meal. It now includes a group of related natural compounds ( menaquinones ), differing in the number of isoprene units in the side chain and in their degree of unsaturation. These quinones also appear to be involved in the electron transport chain and oxidative phosphorylation. 

Cyhexatin [13121 -70-5], tricyclohexylhydroxystannane (147) (mp 195°C), rat oral 540 mg/kg, and fenbutatin oxide [13356-08-6], hexakis-(2-methyl-2-phenylpropyl)distannoxane (148) (mp 138°C), rat oral LD q 2630 mg/kg, are two novel tin acaricides used on deciduous fmits. They are inhibitors of oxidative phosphorylation. 

Sulflutamid or A/-ethylpetfluotoctanesulfonamide [4151 -50-2] CgF yS02NHC2H, is a slow-acting stomach poison used in baits for the control of ants and cockroaches. It acts as an uncoupler of oxidative phosphorylation. 

Phosphorus. Eighty-five percent of the phosphoms, the second most abundant element in the human body, is located in bones and teeth (24,35). Whereas there is constant exchange of calcium and phosphoms between bones and blood, there is very Httle turnover in teeth (25). The Ca P ratio in bones is constant at about 2 1. Every tissue and cell contains phosphoms, generally as a salt or ester of mono-, di-, or tribasic phosphoric acid, as phosphoHpids, or as phosphorylated sugars (24). Phosphoms is involved in a large number and wide variety of metaboHc functions. Examples are carbohydrate metaboHsm (36,37), adenosine triphosphate (ATP) from fatty acid metaboHsm (38), and oxidative phosphorylation (36,39). Common food sources rich in phosphoms are Hsted in Table 5 (see also Phosphorus compounds). 

Two and twelve moles of ATP are produced, respectively, per mole of glucose consumed in the glycolytic pathway and each turn of the Krebs (citrate) cycle. In fat metaboHsm, many high energy bonds are produced per mole of fatty ester oxidized. Eor example, 129 high energy phosphate bonds are produced per mole of palmitate. Oxidative phosphorylation has a remarkable 75% efficiency. Three moles of ATP are utilized per transfer of two electrons, compared to the theoretical four. The process occurs via a series of reactions involving flavoproteins, quinones such as coenzyme Q, and cytochromes. 

Aminophenol is a selective nephrotoxic agent and intermpts proximal tubular function (121,122). Disagreement exists concerning the nephrotoxity of the other isomers although they are not as potent as 4-aminophenol (123,124). Respiration, oxidative phosphorylation, and ATPase activity are inhibited in rat kidney mitochondria (125). The aminophenols and their derivatives are inhibitors of 5-Hpoxygenase (126) and prostaglandin synthetase... 

The importance of quinones with unsaturated side chains in respiratory, photosynthetic, blood-clotting, and oxidative phosphorylation processes has stimulated much research in synthetic methods. The important alkyl- or polyisoprenyltin reagents, eg, (71) or (72), illustrate significant conversions of 2,3-dimethoxy-5-methyl-l,4-ben2oquinone [605-94-7] (73) to 75% (74) [727-81-1] and 94% (75) [4370-61-0] (71—73). 

Bithionol interferes with the neuromuscular physiology of helminths, impairs egg formation, and may cause defects in the protective cuticle covering the worm. At the biochemical level, the oxidative phosphorylation of the worm is inhibited. 

The modes of action for niclosamide are interference with respiration and blockade of glucose uptake. It uncouples oxidative phosphorylation in both mammalian and taenioid mitochondria (22,23), inhibiting the anaerobic incorporation of inorganic phosphate into adenosine triphosphate (ATP). Tapeworms are very sensitive to niclosamide because they depend on the anaerobic metaboHsm of carbohydrates as their major source of energy. Niclosamide has selective toxicity for the parasites as compared with the host because Httle niclosamide is absorbed from the gastrointestinal tract. Adverse effects are uncommon, except for occasional gastrointestinal upset. 

ELAVOPROTEINS. Flavin is an essential substance for the activity of a number of important oxidoreductases. We discuss the chemistry of flavin and its derivatives, FMN and FAD, in the chapter on electron transport and oxidative phosphorylation (Chapter 21). 

The combustion of the acetyl groups of acetyl-CoA by the citric acid cycle and oxidative phosphorylation to produce COg and HgO represents stage 3 of catabolism. The end products of the citric acid cycle, COg and HgO, are the ultimate waste products of aerobic catabolism. As we shall see in Chapter 20, the oxidation of acetyl-CoA during stage 3 metabolism generates most of the energy produced by the cell. 

Although the interior of a prokaryotic cell is not subdivided into compartments by internal membranes, the cell still shows some segregation of metabolism. For example, certain metabolic pathways, such as phospholipid synthesis and oxidative phosphorylation, are localized in the plasma membrane. Also, protein biosynthesis is carried out on ribosomes. 

FIGURE 18.16 Compartmentalization of glycolysis, the citric acid cycle, and oxidative phosphorylation. 

Glycolysis and the citric acid cycle (to be discussed in Chapter 20) are coupled via phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an allosteric inhibitor of phosphofructokinase. When the citric acid cycle reaches saturation, glycolysis (which feeds the citric acid cycle under aerobic conditions) slows down. The citric acid cycle directs electrons into the electron transport chain (for the purpose of ATP synthesis in oxidative phosphorylation) and also provides precursor molecules for biosynthetic pathways. Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated. 

FIGURE 20.1 Pyruvate produced hi glycolysis is oxidized in the tricarboxylic acid (TCA) cycle. Electrons liberated in this oxidation flow through the electron transport chain and drive the synthesis of ATP in oxidative phosphorylation. In eukaryotic cells, this overall process occurs in mitochondria. 

All six carbons of glucose are liberated as CO2, and a total of four molecules of ATP are formed thus far in substrate-level phosphorylations. The 12 reduced coenzymes produced up to this point can eventually produce a maximum of 34 molecules of ATP in the electron transport and oxidative phosphorylation pathways. A stoichiometric relationship for these subsequent processes is 1... 

Thus, a total of 3 ATP per NADH and 2 ATP per FADHa rnay be produced through the processes of electron transport and oxidative phosphorylation. 

Wall Piece IV (1985), a kinetic sculpture by George Rhoads. This complex meehanieal art form can be viewed as a metaphor for the molecular apparatus underlying electron transport and ATP synthesis by oxidative phosphorylation. (1985 ty George Rhoaeh)... 

Whereas ATP made in glycolysis and the TCA cycle is the result of substrate-level phosphorylation, NADH-dependent ATP synthesis is the result of oxidative phosphorylation. Electrons stored in the form of the reduced coenzymes, NADH or [FADHa], are passed through an elaborate and highly orga-... 

Electron Transport and Oxidative Phosphorylation Are Memhrane-Associated Processes... 

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