How is ATP produced without mitochondria?

3. OXIDATIVE PHOSPHORYLATION

Oxidative phosphorylation occurs in the mitochondria of all animal and plant tissues, and is a coupled process between the oxidation of substrates and production of ATP. As the Kreb's cycle runs, hydrogen ions (or electrons) are carried by the two carrier molecules NAD or FAD to the electron transport pumps. The protons are pumped to the intermembrane region where they accumulate in a high enough concentration to phosphorylate the ADP to ATP.

According to the chemiosmotic coupling hypothesis, ATP synthesis decreases proton electrochemical gradient hence stimulates the respiratory chain to pump more protons across the mitochondrial inner membrane and maintaine the gradient. However, electron supply to the respiratory chain also affects respiration and ATP synthesis. For example, calsium stimulates mitochondrial matrix dehydrogenase, and increases the electron supply to the respiratory chain and hence the rate of respiration and ATP synthesis [36].

Control of respiration and ATP synthesis shift as the metabolic state of the mitochondria changes. In state 4 of mitochondria, respiration is low, proton electrochemical gradient is high, and there is no ATP synthesis. However, in state 3 respiration is high, proton electrochmical gradient is lowered, and ATP synthesis is high. In an isolated mitochondria, the control over state 4 respiration is mainly due to the proton leak through the mitochondrial inner membrane. This type of control decreases as mitochondria change from the state 4 to state 3, and the control by the adenine nucleotide and the dicarboxylate carriers, cytochrome oxidase, the ATP utilizing reactions, and transport activities increase. Therefore in state 3, most of the control is due to respiratory chain and substrate transport [36].

It is generally assumed that 2,4-dinitrophenol acts as protonophores, and carries the proton by increasing the proton conductance of the inner membrane. This regulates coupling efficiency between the ATP synthesis and the respiratory rate. According to the chemiosmotic theory, the proton pump is connected by a proton electrochemical potential difference between intermembrane space and matrix. The electrons are carried from complex to complex by ubiquinone and cycochrome c. The ATP synthase uses the proton gradient to form ATP from ADP and phosphate. The cristae house and organize the electron transport chain and the ATP pumps. A terminal enzyme in the respiratory chain, cytochrome c oxidase, reduces oxygen to water. Thus, each compartment in the mitochondrion is specialized for one phase of these reactions (Fig. 3).

Without oxygen only 4 molecules of ATP energy packets are produced for each glucose molecule (in glycolysis). Oxidative phosphorylation produces 24–28 ATP molecules from the Kreb's cycle from one molecule of glucose converted into pyruvate.

Two theoretical approaches applied to the oxidative phosphorylation are metabolic control analysis and nonequilibrium thermodynamics. These approaches are helpful for quantitative description and understanding of control and regulation of the oxidative phosphorylation [36, 46]. For example metabolic control theory can provide a quantitative decription for the microbial growth [79].

The application of nonequilibrium thermodynamics is one of the early attempts to fornulate oxidative phosphorylation in a quantitative manner. This application assumes a linear flow-force relationships for the oxidation and phosphorylation flows. Such a linear dependence between the flows and thermodynamic forces has been established by measurements during state 4 to state 3 transistion as a linear part of a more general sigmodial relationship. Nonequilibrium thermodynamics has proved to be useful especialy in describing the energetics aspects of oxidative ATP production and the transport of substrates coupled to ATP hydrolysis.

The thermodynamics approach is an effort of a unified description of a system. However, living systems are complex, and it is not realistic to assume that a simple mathematical formalism can yield a complete description of such complex and coupled systems. Beside thermodynamics tool, mathematical tools such as metabolic control analysis can also be useful to interpret the measured properties of oxidative phosphorylation. A kinetic model of oxidative phosphorylation may not be fully and definetely completed, because of the several assumptions and simplifications associated with it. A proper kinetic approach can relate macroscopic behavior to microscopic properties, and hence allows a deeper insight into the mechanisms related to the control and regulation of oxidative phosphorylation; it may provide a modeling procedure and methodological approach to describe dynamic and stationary properties of energy coupling in membranes [104]. The application of metabolic control analysis to mitochondria may describe how control is distributed throughout the mitochondria, but does not predict how the systems are regulated, which may be improved by developing of a regulation analysis [36].

The linear nonequilibrium thermodynamic theory and quasi-linear flow-force relations may be useful for decribing a simplified example of oxidative phosphorylation [69], and the dissipation function is given by

Here the subscripts P, H, and O refer to the phosphorylation, H+ flow, and substrate oxidation respectively, and ΔμH=Δ μHi−ΔμHo. We consider only steady states. The dissipation function can be transformed as

Where Aex is the external affinity. When the interior of the mitochondrion is in a stationary state, it suffices to measure the changes in the external solution only.

From Eq. (2) the appropriate phenomenological equations in terms of the resistance formulations are expressed by

For the oxidative phosphorylation we have the three degrees of couplings, qPH, qOH, and qPO, . If Aoex is kept constant and ΔμH is not controlled, JH = 0 in the stationary state, which is also called the static head, and Eqs. (3) and (5) become

Consequently, the degree of coupling is given by.

When we have the level flow, the force vanishes, ΔμH = 0, and Eqs. (3) to (5) give

(9)APex=KP(1−qP H2)JP−(KPKO)1/2 (qPO+qPHqOH)JO

(10)AOex=−(KPKO) 1/2(qPO+qPHqOH )JP+KO(1−qOH2)JO

where the term q is given by

(11)q=qPO+qPHqOH(1−qPH2)(1−qOH 2)

When the rate of performance of electroosmotic work is appreciable, we can define the effectiveness of energy conversion, which is expressed by

where Xp is the force for proton transportation.

It is also useful to consider the force developed per given rate of expendeture of metabolic energy, which is caled the efficiacy of force

9.7.4 Fermentative Pathways

When the oxidative phosphorylation is inactive (due to the absence of oxygen, or lack of some of the necessary proteins), pyruvate is not oxidized in the TCA cycle since that would lead to an accumulation of NADH inside the cells. In this situation, NADH is oxidized with simultaneous reduction of pyruvate to acetate, lactic acid, or ethanol. These processes are collectively called fermentative metabolism. Fermentative metabolism is not the same in all microorganisms, but there are many similarities.

Bacteria can regenerate all NAD+ by reduction of pyruvate to lactic acid, see Fig. 9.29A and B. They can also regenerate all NAD+ by formation of ethanol in the so-called mixed acid fermentation pathway, for which the entry point is the compound Acetyl-CoA. CoA is a cofactor with a free –SH group that can be acetylated to CH3CO–S–, as is illustrated in Fig. 9.24, either directly from acetate or by capturing two of the carbon atoms of pyruvate with the last carbon atom liberated as carbon dioxide or formic acid, HCOOH. Lactic acid bacteria (Fig. 9.29B) have both pathways for the conversion of one pyruvate to acetyl-CoA, whereas E. coli only has the pyruvate formate lyase catalyzed reaction. In yeast, the fermentative pathway does not proceed via acetyl-CoA, but instead by decarboxylation of pyruvate to acetaldehyde. From acetate, cytosolic acetyl-CoA may be synthesized, and this serves as a precursor for fatty acid biosynthesis; whereas the mitochondrial acetyl-CoA, which is formed directly from pyruvate, serves as an entry point to the TCA cycle. In yeast, the primary metabolic product is ethanol, but even with respiratory growth, where complete reoxidation of NADH is possible through oxidative phosphorylation, the pyruvate dehydrogenase complex (Fig. 9.29C), which catalyzes the direct conversion of pyruvate to acetyl-CoA, may be bypassed as indicated. Above a certain glucose uptake rate, the respiratory capacity becomes limiting; and this leads to overflow in the bypass, and consequently ethanol is formed. This overflow metabolism is traditionally referred to as the Crabtree effect.

Acetyl-CoA can be regarded as an activated form of acetic acid as it can be converted to acetic acid; see Fig. 9.29A and B. As seen in the last step of the acetic acid formation, an ATP is released, thereby doubling the ATP yield through the catabolism of glucose from 2 to 4 ATP per glucose molecule. This is the reason why bacteria use the mixed acid pathways at very low glucose fluxes. In order to obtain a complete regeneration of NAD+, the flow of carbon to the metabolic end products of formic acid, ethanol, and acetic acid must, however, be balanced.

Finally, it should be noted that the pathways shown in Fig. 9.29 are, by necessity, quite simplified. Thus E. coli succinate may be an end product. Furthermore, in some bacteria alternative pathways from pyruvate to other end products, such as butanol (together with butyric acid and acetone) or to 2,3-butanediol (together with acetoin), may be active.

Chloroplasts

Whereas in oxidative phosphorylation electrons are transferred from a low-energy electron donor (e.g., NADH) to an acceptor (e.g., O2) through an electron-transport chain, in photophosphorylation, which takes place in chloroplasts, the energy of sunlight is used to create a high-energy electron donor and an electron acceptor. Electrons are then transferred from the donor to the acceptor through another electron-transport chain.

In the thylakoid membranes of choloroplasts two kinds of photophosphorylation take place: cyclic and non-cyclic photophosphorylation.

In cyclic electron flow, the electron begins in a pigment complex called photosystem I, passes from the primary acceptor to ferredoxin, then to cytochrome b6f (a similar complex to that found in mitochondria), and then to plastocyanin before returning to chlorophyll. This transport chain produces a proton-motive force, pumping H+ ions across the membrane; this produces a concentration gradient that can be used to power ATP synthase during chemiosmosis. This pathway is known as cyclic photophosphorylation, and produces neither O2 nor NADPH. Unlike non-cyclic photophosphorylation, NADP+ does not accept the electrons; they are instead sent back to photosystem I.

The other pathway, noncyclic photophosphorylation, is a two-stage process involving two different chlorophyll photosystems. First, a water molecule is broken down into 2H+ + 1/2 O2 + 2e− by a process of photolysis. The two electrons from the water molecule are kept in photosystem II, while the 2H+ and 1/2O2 are left out for further use. Then a photon is absorbed by chlorophyll pigments that surround the reaction center of the photosystem. The light excites the electrons of each pigment, causing a chain reaction that eventually transfers energy to the core of photosystem II, exciting the two electrons that are transferred 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, which takes the 2e− from pheophytin and two H+ from the stroma, and forms PQH2, which later is broken into PQ; the 2e− is released to cyt b6f complex and the two H+ are released into the thylakoid lumen. The electrons then pass through cyt b6 and cyt f. Then they are passed to plastocyanin, providing the energy for protons to be pumped into the thylakoid space. This generates a gradient, making H+ ions flow back into the stroma of the chloroplast, thus providing the energy for the regeneration of ATP.

The photosystem II complex replaces its lost electrons from an external source; however, the two other electrons are not returned to photosystem II as they would be in the analogous cyclic pathway. Rather, the still-excited electrons are transferred to a photosystem I complex, which boosts their energy to a higher level using a second solar photon. The highly excited electrons are transferred to the acceptor molecule, but this time they are passed on to an enzyme called ferredoxin-NADP+ reductase, which uses them to reduce NADP+ to NADPH.

This consumes protons produced by the splitting of water, leading to a net production of 1/2O2, ATP and NADPH+H+ with the consumption of solar photons and water.

The concentration of NADPH in the chloroplast may help regulate which pathway electrons take through the light reactions. When the chloroplast runs low on ATP for the Calvin cycle, NADPH will accumulate and the plant may shift from noncyclic to cyclic electron flow.

11.3.3 Phosphorylation

In eukaryotes, oxidative phosphorylation occurs in mitochondria, while photophosphorylation occurs in chloroplasts to produce ATP. Oxidative phosphorylation involves the reduction of O2 to H2O with electrons donated by NADH and FADH2 in all aerobic organisms (Fig. 11.2). After, carbon fuels (nutrients) are oxidized in the citric acid cycle, electrons with electron-motive force is converted into a proton-motive force. Photophosphorylation involves the oxidation of H2O to O2, with NADP+ as electron acceptor. Therefore, the oxidation and the phosphorylation of ADP are coupled by a proton gradient across the membrane. In both organelles mitochondria and chloroplast, electron transport chains pump protons across a membrane from a low proton concentration region to one of high concentration. The protons flow back from intermembrane to the matrix in mitochondria, and from thylakoid to stroma in chloroplast through ATP synthase to drive the synthesis of adenosine triphosphate. Therefore, the adenosine triphosphate is produced within the matrix of mitochondria and within the stroma of chloroplast.

As the tricarboxylic acid cycle runs, hydrogen ions (or electrons) are carried by the two carrier molecules NAD or FAD to the electron transport pumps. Energy released by the electron transfer processes pumps the protons to the intermembrane region, where they accumulate in a high enough concentration to phosphorylate the ADP to ATP. The overall process is called oxidative phosphorylation. The cristae have the major coupling factors F1 (a hydrophilic protein) and Fo (a hydrophobic lipoprotein complex). F1 and Fo together comprise the ATPase (also called ATP synthase) complex activated by Mg2+. Fo forms a proton translocation pathway and F1 is a catalytic sector. ATP synthesis by FoF1 consists of three step: (1) proton translocation through Fo, (2) conformation transmission to F1, and (3) ATP synthesis in the β unit. The rotation of a subunit assembly is an essential feature of the mechanisms of ATP synthesis and can be regarded as a molecular motor. ATP produced in the mitochondria exits to the cytosol to be hydrolyzed within the cell functions such as molecular pumps. Adenosine diphosphate and inorganic phosphate Pi, produced from ATP hydrolysis in the cytosol reenter the mitochondria to be converted again to ATP.

ATPase can catalyze the synthesis and the hydrolysis of ATP, depending on the change of electrochemical potential of proton Δμ˜H. The ratio of ATP production to oxygen consumption (P/O) can vary according to various physiological processes: (i) maximizing ATP production, (ii) maximizing the cellular phosphate potential, (iii) minimizing the cost of production, and (iv) a combination of these three processes.

The values of P/O change within the range of 1–3, and characteristic of the substrate undergoing oxidation and characteristic of the organ's physiological role. In the case of excess oxygen and inorganic phosphate, the respiratory activity of the mitochondria is controlled by the amount of ADP available. In the controlled state called state 4, the amount of ADP is low. With the addition of ADP, the respiratory rate increases sharply; this active state is called state 3. The ratio of the respiratory rates of state 3 to state 4 is called the respiratory control index.

The control of the respiration process and ATP synthesis shifts as the metabolic state of the mitochondria changes. In an isolated mitochondrion, control over the respiration process in state 4 is due to the proton leak through the mitochondrial inner membrane. This type of control decreases from state 4 to state 3, while the control by the adenine nucleotide and the dicarboxylate carriers, cytochrome oxidase, increases. ATP-utilizing reactions and transport activities also increase. Therefore, in state 3, most of the control is due to respiratory chain and substrate transport.

According to the chemiosmotic coupling hypothesis, ATP synthesis decreases the proton electrochemical gradient and hence stimulates the respiratory chain to pump more protons across the mitochondrial inner membrane and maintain the gradient. However, electron supply to the respiratory chain also affects respiration and ATP synthesis. For example, calcium stimulates mitochondrial matrix dehydrogenase, and increases the electron supply to the respiratory chain and hence the rate of respiration and ATP synthesis.

1 INTRODUCTION

In the past decade, a vast amount of literature on mitochondrial cation transport has appeared. A number of excellent reviews have covered in detail the ion translocating properties of mitochondria and of submitochondrial particles obtained from intact mitochondria by various treatments (Lehninger et al., 1967; Greville, 1969; Chance and Montal, 1972; Van Dam and Meijer, 1971). Most of the work is done within the framework of the study of oxidative phosphorylation and mitochondrial energy metabolism; therefore, only a general description of mitochondrial ion transport will be given in this review.

Since the early observations that mitochondria contain and are able to retain cations (Slater and Cleland, 1953; Bartley and Davies, 1954), a considerable increase in the knowledge of ion transport has occurred. Cation uptake by mitochondria is an energy-requiring process. Therefore, in analogy with the various hypotheses of oxidative phosphorylation (see Section III,F), two possibilities of energy-driven cation transport are currently being considered (cf. Greville, 1969).

II.C ATP Synthesis

ATP synthesis in chloroplasts is called photophosphorylation and is similar to oxidative phosphorylation in mitochondria. The light-driven transport of electrons from water to NADP+ is coupled to the translocation of protons from the stroma across the thylakoid membrane (the green, energy-converting membrane) into the lumen. Electron transport from Q− to P700+ is exergonic. Part of the energy released by electron transport is conserved by the formation of an electrochemical proton gradient. The cytochrome b6f complex of chloroplasts functions not only in electron transport, but also in proton translocation.

The active site of the oxygen-evolving enzyme is arranged so that the protons formed during water oxidation are released into the thylakoid lumen. These protons contribute to the electrochemical proton potential. The thylakoid membrane contains a protein that functions to transport Cl− across the membrane. Proton accumulation in the thylakoid lumen is electrically balanced in large part by Cl− uptake. As a result, thylakoids accumulate HCl and the membrane potential across the membrane is low. The pH inside the lumen during steady-state photosynthesis is about 5.0.

One of the earliest experiments that supported the hypothesis that ATP synthesis and electron transport were linked by the electrochemical proton potential was carried out with isolated thylakoid membranes. Thylakoid membranes were placed in a buffer at pH 4.0 and after a few seconds the pH was rapidly increased to 8.0, which resulted in the formation of a proton activity gradient. This artificially formed gradient was shown to drive the synthesis of ATP from ADP and Pi. The experiments were carried out in the dark so that the possibility that electron transport contributed to the ATP synthesis was excluded. Thus, a proton activity gradient was proven capable of driving ATP synthesis.

The thylakoid membrane enzyme that couples ATP synthesis to the flow of protons down their electrochemical gradient is called the chloroplast ATP synthase (see Fig. 10). This enzyme has remarkable similarities to ATP synthases in mitochondria and certain bacteria. For example, the β subunits of the chloroplast ATP synthase have 76% amino acid sequence identity with the β subunits of the ATP synthase of the bacterium E. coli.

The reaction catalyzed by ATP synthases is

(11)nHa++ADP+Pi+H+→nHb++ATP+ H2O,

where n is the number of protons translocated per ATP synthesized, probably three or four, and a and b refer to the opposite sides of the coupling membrane. Provided the electrochemical proton potential is high, the reaction is poised in the direction of ATP synthesis. In principle, when the proton potential is low, ATP synthases should hydrolyze ATP and cause the pumping of protons across the membrane in the direction opposite that which occurs during ATP synthesis. ATP-dependent proton transport by the ATP synthase is of physiological significance in E. coli under anaerobic conditions in that it generates the electrochemical proton potential across the plasma membrane of the bacterium. This potential is used for the active uptake of some carbohydrates and amino acids.

In contrast, ATP hydrolysis by the chloroplast ATP synthase in the dark has no physiological role and would be wasteful. In fact, the rate of ATP hydrolysis by the ATP synthase in thylakoids in the dark is less than 1% of the rate of ATP synthesis in the light. Remarkably, within 10–20 msec after the initiation of illumination, ATP synthesis reaches its steady-state rate. Thus, the activity of the chloroplast ATP synthase is switched on in the light and off in the dark. In addition to being the driving force for ATP synthesis, the electrochemical proton potential is involved in switching the enzyme on. Structural perturbations of the enzyme induced by the proton potential overcome inhibitory interactions with bound ADP as well as with a polypeptide subunit of the synthase. An additional regulatory mechanism that is unique to the chloroplast ATP synthase is reductive activation. Reduction of a disulfide bond in a subunit of the chloroplast ATP synthase to a dithiol enhances the rate of ATP synthesis, especially at physiological values of the proton potential. The electrons for this reduction are derived from the chloroplast electron transport chain.

Action of Anacardic acids towards Cell components and Membranes

Anacardic acids have been shown to have an uncoupling effect on the oxidative phosphorylation in rat liver mitochondria, the energy-responsible components of cells [294] similar to the classical uncoupler, 2,4-dinitrophenol. (15:1)-Anacardic acid was the most effective of the four acids, while salicylic acid had a very weak effect. Changes in liposomal membrane potential and pH gradient are anacardic acid-mediated and are considered to provide evidence for their having a unique function as both negative charge carriers and also ‘proton carriers’ that dissipate the transmembrane proton gradient formed [295]. It seems likely that these findings might be explained by the molecular changes that occur with with reduction of pH, namely, dianion → monoanion → intramolecular hydrogen-bonded structure. or at least by the two latter states.

In the marine environment there has been the first reported occurrence of so-called antifouling (namely, anticrustaceous activity) by (15:0)-anacardic acid. Thus bioassay-directed fractionation of the dichloromethane extract from twigs of Ozoroa insignis, containing the phenolic lipid, was used with larvae from Artemia salina as a model to investigate the activity [296].

Publisher Summary

This chapter describes the structure and function of ATP synthase. ATP is synthesized through oxidative phosphorylations by an ATP-synthase complex coupled to the respiratory chain. All ATP-synthase complexes producing great amounts of ATP in living organisms are coupled to chains of electron transporters integrated in membranes or in lipid–protein lamellar structures, which are named transducing membranes. In each case, the transporter chain is reduced at one end by a flow of electrons and protons, and the ATP synthesis occurs during the reoxidation process of the chain, using the liberated redox energy. It has been stressed in various studies that ATP-synthases exhibit very striking similarities of structure and properties. By the use of various techniques, ATP-synthase has been either isolated as a partially purified oligomycine-sensitive ATPase (OS-ATPase) or progressively dissociated in its various sectors. A small protein inhibitor has been found associated to ATP-synthases. It can be dissociated from the membrane by an alkali treatment followed by a Sephadex G-50 chromatography.

1.1.2.2.3 Cytochrome

Cytochromes are iron-binding proteins that play an important role in intracellular electron transfer and oxidative phosphorylation. In S. oneidensis MR 1, c-type cytochromes present on the cell wall facilitate the transfer of electrons from the bacterium to the electrode. The cytochromes are multiheme-binding cell-bound proteins. The c-type cytochrome forms a multilayer biofilm that produces a higher potential than a monolayered biofilm. The cyclic voltammetry (CV) analysis of biofilm showed active redox peaks, confirming the role of cytochromes in electron shuttling. The mutants deficient in cytochrome synthesis were incapable of electron mediation. Inoue et al. showed that c-type cytochrome omcZ is essential for electron transfer in G. sulfurreducens. Mutants lacking the gene for omcZ showed poor electron conduction and current production [20]. Flavins such as FAD and riboflavins in S. oneidensis have been shown to be electron mediators. The redox property and ability to transfer electrons between the bacterial cell and electrode were analyzed using CV and showed the role of riboflavin in electron shuttling and metal reduction in S. oneidensis.

The electroactive bacteria in MFCs may use more than one mechanism of electron transfer. More often the direct electron transfer occurs with the cumulative effects of a biofilm, pili, and cytochromes for exoelectron transfer in MFC. Several studies have reported on the role of pili in biofilm formation, electron mediators present in the extracellular polysaccharide matrix of biofilm, and the outer membrane cytochromes for electrode and metal reduction.

Necrosis

Two phenomena consistently characterize irreversible injury. The first is the inability to reverse mitochondrial dysfunction (lack of oxidative phosphorylation and ATP generation) even upon restoration of oxygen; the second is the development of profound disturbances in membrane function. Massive calcium influx into the cell occurs, particularly if ischemic tissue is reperfused after the point of irreversible injury, with broad activation of calcium-dependent catabolic enzymes. In addition, proteins, essential coenzymes, and ribonucleic acids seep out through the newly permeable membranes, and the cells also lose metabolites vital for the reconstitution of ATP. Injury to the lysosomal membranes results in leakage of their enzymes into the cytoplasm; the catabolic enzymes are activated in the reduced intracellular pH of the ischemic cell, and will further degrade cytoplasmic and nuclear components.

Since necrotic tissue is a potential nidus for secondary infections, the body rapidly mobilizes inflammatory cells to clean up the resulting debris, and initiates the process of either rebuilding the dead tissue or laying down a fibrous scar (see Chapter II.1.5). As mentioned previously, the recruited inflammation can in itself be a cause of further local injury. Moreover, having to replace a specialized tissue with a non-functional matrix scar is also a less than optimal outcome.