Coupled ATP formation

As with chloroplasts, many questions concerning electron flow and the coupled ATP formation in mitochondria remain unanswered. The first part of the mitochondrial electron transfer chain has a number of two-electron carriers (NAD+, FMN, and ubiquinone) that interact with the cytochromes (one-electron carriers). In this regard, the reduction of O2 apparently involves four electrons coming sequentially from the same Cyt a3. Of... 

Unfortunately the answer to our third question, that of the mechanism of coupling, is the subject of heated controversy between various research workers and undoubtedly the exact nature of the gears that couple ATP formation to electron transport are still to be worked out. However, any hypothesis has to account for a number of observed experimental results, the most important of which are ... 

However the formation ofXY will not proceed spontaneously because the free energy of the product PCY) exceeds the free energy of the substrates (X and Y). We refer to the formation of XV as being an unfavorable process because, for Equation (4), AG > 0. Cells can form the XY they need only by coupling its formation to a reaction, such as the breakdown of ATP, that provides the energy required to build the chemical bonds that hold X and Y together. This process is shown in the coupled reaction below ... 

Condensation of CO2, ammonia, and ATP to form carbamoyl phosphate is catalyzed by mitochondrial carbamoyl phosphate synthase I (reaction 1, Figure 29-9). A cytosolic form of this enzyme, carbamoyl phosphate synthase II, uses glutamine rather than ammonia as the nitrogen donor and functions in pyrimidine biosynthesis (see Chapter 34). Carbamoyl phosphate synthase I, the rate-hmiting enzyme of the urea cycle, is active only in the presence of its allosteric activator JV-acetylglutamate, which enhances the affinity of the synthase for ATP. Formation of carbamoyl phosphate requires 2 mol of ATP, one of which serves as a phosphate donor. Conversion of the second ATP to AMP and pyrophosphate, coupled to the hydrolysis of pyrophosphate to orthophosphate, provides the driving... 

The spatial separation between the components of the electron transport chain and the site of ATP synthesis was incompatible with simple interpretations of the chemical coupling hypothesis. In 1964, Paul Boyer suggested that conformational changes in components in the electron transport system consequent to electron transfer might be coupled to ATP formation, the conformational coupling hypothesis. No evidence for direct association has been forthcoming but conformational changes in the subunits of the FI particle are now included in the current mechanism for oxidative phosphorylation. 

Once the phosphate ester is hydrolysed, there is an immediate rapid tautomerism to the keto form, which becomes the driving force for the metabolic transformation of phosphoenolpyruvic acid into pyruvic acid, and explains the large negative free energy change in the transformation. This energy release is coupled to ATP formation (see Box 7.25). 

Oxidizible substrates from glycolysis, fatty acid or protein catabolism enter the mitochondrion in the form of acetyl-CoA, or as other intermediaries of the Krebs cycle, which resides within the mitochondrial matrix. Reducing equivalents in the form of NADH and FADH pass electrons to complex I (NADH-ubiquinone oxidore-ductase) or complex II (succinate dehydrogenase) of the electron transport chain, respectively. Electrons pass from complex I and II to complex III (ubiquinol-cyto-chrome c oxidoreductase) and then to complex IV (cytochrome c oxidase) which accumulates four electrons and then tetravalently reduces O2 to water. Protons are pumped into the inner membrane space at complexes I, II and IV and then diffuse down their concentration gradient through complex V (FoFi-ATPase), where their potential energy is captured in the form of ATP. In this way, ATP formation is coupled to electron transport and the formation of water, a process termed oxidative phosphorylation (OXPHOS). 

ATP Formation Coupled to Glycolysis During glycolysis some of the energy of the glucose molecule is conserved in ATP, while much remains in the product, pyruvate. The overall equation for glycolysis is... 

A requirement for all fermentations is the existence of a mechanism for coupling ATP synthesis to the fermentation reactions. In the lactic acid and ethanol fermentations this coupling mechanism consists of the formation of the intermediate 1,3-bisphosphoglycerate by the glyceraldehyde 3-phosphate dehydrogenase (Fig. 10-3, step a). This intermediate contains parts of both the products ATP and lactate or ethanol. 

Like mitochondria, chloroplasts (when illuminated) pump protons across their membranes (Fig. 23-18). However, while mitochondria pump protons to the outside, the protons accumulate on the inside of the thylakoids. The ATP synthase heads of coupling factor CEj are found on the outside of the thylakoids, facing the stromal matrix, while those of F, lie on the insides of mitochondrial membranes. However, the same mechanism of ATP formation is used in both chloroplasts and mitochondria (Chapter 18). 

All of these reactions release energy. In biological oxidations much of the energy is utilized to form ATP from ADP and inorganic phosphate (Section 15-5F). That is to say, electron-transfer reactions are coupled with ATP formation. The overall process is called oxidative phosphorylation. 

Figure 20-7 Simplified representation of the photoreactions in photosynthesis. The oxidation of water is linked to the reduction of NADP by an electron-transport chain (dashed line) that is coupled to ATP formation (photophosphorylation).

Oxidative phosphorylation resembles photophosphorylation, discussed in Section 20-9, in that electron transport in photosynthesis also is coupled with ATP formation. 

Electron Transfer Is Coupled to ATP Formation at Three Sites... 

Warburg showed how ATP formation is coupled to the dehydrogenation of glyceraldehyde-3-phosphate. 

Glutamine synthetase catalyzes the incorporation of ammonia into glutamine, deriving energy from the hydrolysis of ATP (Fig. 3b). This enzyme is named a synthetase, rather than a synthase, because the reaction couples bond formation with the hydrolysis of ATP. In contrast, a synthase does not require ATP. 

Calculate AE 0 and AG° for each redox step, and identify the sites that may couple with phosphorylation (ATP formation) assuming AG° = —30.5 kJ mol 1 for the hydrolysis of ATP to ADP. 

Fig. 14.40. The localized model for ATP formation (in a particle). The particle is illustrated by the box, and the diffusion-controlled paths of e and H+ are shown. The device can be converted into a chemical model of a membrane [see dashed vertical lines around (b)], to give two aqueous phases (a) and (c) not in equilibrium with (b). This becomes the chemosmosis model by releasing all H+ to (c) and all of to (a). These are three models (there are several more) for devices that can use an energized proton to make ATP or to couple other energy-driven processes. (Reprinted from R.J.P. Williams, FEBS Lett. 85 10, Fig. 1, copyright 1978 with permission from Elsevier Science.)...

It is now evident that at least two, and probably more, of the four possible reductive steps are coupled to ATP formation in denitrifying bacteria. As may be expected from thermodynamic consideration, this crucial observation implies an ATP yield per mole of glucose similar to that for normal oxidative metabolism (see Gottschalk, 1979, for literature in this area). 

Figure 5-1. Schematic representation of the three stages of photosynthesis in chloroplasts (1) The absorption of light can excite photosynthetic pigments, leading to the photochemical events in which electrons are donated by special chlorophylls. (2) The elections are then transferred along a series of molecules, causing the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) to become the reduced form (NADPH) ATP formation is coupled to the electron transfer steps. (3) The biochemistry of photosynthesis can proceed in the dark and requires 3 mol of ATP and 2 mol of NADPH per mole of C02 fixed into a carbohydrate, represented in the figure by (CH20).

D. An uncoupler is a compound that decreases the ATP formation coupled to photosynthetic electron flow. When such a compound is added to chloroplasts incubated at a high photon flux density, the O2 evolution rate eventually becomes less than a control without the uncoupler. Explain. 

In Chapter 5 (Section 5.5B), we introduced the various molecules involved with electron transfer in chloroplasts, together with a consideration of the sequence of electron flow between components (Table 5-3). Now that the concept of redox potential has been presented, we will resume our discussion of electron transfer in chloroplasts. We will compare the midpoint redox potentials of the various redox couples not only to help understand the direction of spontaneous electron flow but also to see the important role of light absorption in changing the redox properties of trap chi. Also, we will consider how ATP formation is coupled to electron flow. 

Figure 6-5. Energetics and directionality of the coupling between electron flow and ATP formation in chloroplasts, emphasizing the role played by H+ (see also Fig. 5-19). The02 evolution from H20 and the electron flow via plastoquinones (PQ) and the cytochrome complex (Cyt b6f) lead to H+ accumulation in the lumen of a thylakoid. This H+ can moveback out through a hydrophobic channel (CF0) and another protein factor (CF, which together comprise the ATP synthetase, leading to ATP formation.

We next reconsider the vectorial aspects of proton and electron flow (Figs. 5-19 and 6-5) and examine the associated energetics. We will discuss the structures involved in the coupling of ATP formation to proton flow. We will also consider the stoichiometry of the various flows with respect to the ATP and NADPH requirements of CO2 fixation. 

Coupling between the H+ movements across the thylakoid membranes associated with electron flow and ATP formation occurs via a coupling factor known as an ATP synthetase, which is usually referred to as ATP synthase but also as an ATPase (because it can catalyze the reverse reaction leading to ATP hydrolysis). As illustrated in Figure 6-5, the ATP synthase has two components (1) a five-protein factor that occurs on the stromal side of a thylakoid, which can bind ADP, Pj, and ATP (labeled CFX in Fig. 6-5) and (2) a four-protein factor that is hydrophobic and hence occurs in the thylakoid membrane, through which H+ can pass (labeled CF0).5... 

The activities of chloroplasts and mitochondria are related in various ways (Fig. 6-7). For instance, the O2 evolved by photosynthesis can be consumed during respiration, and the CO2 produced by respiration can be fixed by photosynthesis. Moreover, ATP formation is coupled to electron flow in... 

ATP formation coupled to electron flow in mitochondria is usually called oxidative phosphorylation. Because electron flow involves both reduction and oxidation, more appropriate names are respiratory phosphorylation and respiratory-chain phosphorylation, terminology that is also more consistent with photophosphorylation for ATP formation in photosynthesis. As with photophosphorylation, the mechanism of oxidative phosphorylation is not yet fully understood in molecular terms. Processes like phosphorylation accompanying electron flow are intimately connected with membrane structure, so they are much more difficult to study than are the biochemical reactions taking place in solution. A chemiosmotic coupling mechanism between electron flow and ATP formation in mitochondria is generally accepted, and we will discuss some of its characteristics next. 

ATP formation is coupled to the energetically downhill H+ movement back into the mitochondrial matrix through a hydrophobic protein factor in the inner membrane (F0 see footnote 5) and a protein factor (Fx) about 9 nm in diameter that protrudes from the inner membrane into the matrix (Fig. 6-9). Indeed, subunits of Fx rotate during ATP formation, so this protein structure has been called a rotary motor and has become a model for... 

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