The phosphorylation of ADP to ATP

A small number of metabolic reactions involve direct transfer of phosphate from a phosphorylated substrate onto ADP, forming ATP — substrate-level phosphorylation. Two such reactions are shown in Figure 3.13 — both are reactions in the glycolytic pathway of glucose metabolism (section 5.4.1). Substrate-level phosphorylation is of

Muscle Metabolism And Atp
Figure 3.13 Formation of ATP by substrate level phosphorylation (see also Figure 5.10).

relatively minor importance in ensuring a supply of ATP, although, as discussed in section 5.4.1.2, it becomes important in muscle under conditions of maximum exertion. Normally almost all of the phosphorylation of ADP to ATP occurs in the mitochondria, by the process of oxidative phosphorylation.

3.3.1 Oxidative phosphorylation: the phosphorylation of adp to ATP linked to the oxidation of metabolic fuels

With the exception of glycolysis (section 5.4.1), which is a cytosolic pathway, most of the reactions in the oxidation of metabolic fuels occur inside the mitochondria, and lead to the reduction of nicotinamide nucleotide and flavin coenzymes (section 2.4.1). The reduced coenzymes are then reoxidized, ultimately leading to the reduction of oxygen to water. Within the inner membrane of the mitochondrion (section 3.3.1.2) there is a series of coenzymes that are able to undergo reduction and oxidation. The first coenzyme in the chain is reduced by reaction with NADH, and is then reoxidized by reducing the next coenzyme. In turn, each coenzyme in the chain is reduced by the preceding coenzyme, and then reoxidized by reducing the next one. The final step is the oxidation of a reduced coenzyme by oxygen, resulting in the formation of water.

Experimentally, mitochondrial metabolism is measured using the oxygen electrode, in which the percentage saturation of the buffer with oxygen is measured electrochemically as the mitochondria oxidize substrates and reduce oxygen to water.

Figure 3.14 shows the oxygen electrode traces for mitochondria incubated with varying amounts of ADP, and a super-abundant amount of malate. As more ADP is provided, so there is more oxidation of substrate, and hence more consumption of mitochondria + substrate added

ADP added

time

Figure 3.14 Oxygen consumption by mitochondria incubated with malate and varying amounts of ADP.

Figure 3.14 Oxygen consumption by mitochondria incubated with malate and varying amounts of ADP.

time

Figure 3.15 Oxygen consumption by mitochondria incubated with malate or succinate and a constant amount of ADP.

oxygen. This illustrates the tight coupling between the oxidation of metabolic fuels and availability of ADP shown in Figure 3.2.

Figure 3.15 shows the oxygen electrode traces for incubation of mitochondria with a limiting amount of ADP and:

1 malate, which reduces NAD+ to NADH;

2 succinate, which reduces a flavin coenzyme, then ubiquinone.

The stepwise oxidation of NADH and reduction of oxygen to water is obligatorily linked to the phosphorylation of ADP to ATP. Approximately 3 mol of ATP is formed for each mole of NADH that is oxidized. Flavoproteins reduce ubiquinone, which is an intermediate coenzyme in the chain, and approximately 2 mol of ADP is phosphorylated to ATP for each mole of reduced flavoprotein that is oxidized.

This means that 2 mol of ADP is required for the oxidation of a substrate such as succinate, but 3 mol of ADP is required for the oxidation of malate. Therefore, the oxidation of succinate will consume more oxygen when ADP is limiting than does the oxidation of malate. This is usually expressed as the ratio of phosphate to oxygen consumed in the reaction; the P/O ratio is approximately 3 for malate and approximately 2 for succinate.

3.3.1.1 The mitochondrion

Mitochondria are intracellular organelles with a double-membrane structure. Both the number and size of mitochondria vary in different cells — for example, a liver cell contains some 800 mitochondria, a renal tubule cell some 300 and a sperm about 20. The outer mitochondrial membrane is permeable to a great many substrates, while the inner membrane provides a barrier to regulate the uptake of substrates and output of products (see, for example, the regulation of palmitoyl CoA uptake into the mitochondrion for oxidation in section 5.5.1).

The inner mitochondrial membrane forms the cristae, which are paddle-shaped, double-membrane structures that protrude from the inner membrane into the matrix, as shown in Figure 3.16. The crista membrane is continuous with the inner mitochondrial membrane, and the internal space of the crista is contiguous with the inter-membrane space. However, there is only a relatively narrow stalk connecting the crista to the inter-membrane space, so that the crista space is effectively separate from, albeit communicating with, the inter-membrane space.

The five compartments of the mitochondrion have a range of specialized functions:

1 The outer membrane contains the enzymes that are responsible for the desaturation and elongation of fatty acids synthesized in the cytosol (section 5.6.1.1), the enzymes for triacylglycerol synthesis from fatty acids (section 5.6.1.2) and phospholipases that catalyse the hydrolysis of phospholipids (section 4.3.1.2).

2 The inter-membrane space contains enzymes involved in nucleotide metabolism, transamination of amino acids (section 9.3.1.2) and a variety of kinases.

Mitochondrion
Figure 3.16 The membranes of the mitochondrion and infolding to form mitochondrial cristae.

3 The inner membrane regulates the uptake of substrates into the matrix for oxidation. There is also a transport protein in the mitochondrial inner membrane that transports ADP into the matrix to undergo phosphorylation only in exchange for ATP being transported out to the cytosol.

4 The membrane of the cristae contains the coenzymes associated with electron transport, the oxidation of reduced coenzymes, and the reduction of oxygen to water (section 3.3.1.2). The primary particles on the matrix surface of the cristae contain the enzyme that catalyses the phosphorylation of ADP to ATP (section 3.3.1.3).

5 The mitochondrial matrix contains the enzymes concerned with the oxidation of fatty acids (section 5.5.2), the citric acid cycle (section 5.4.4), a variety of other oxidases and dehydrogenases, the enzymes for mitochondrial replication and the DNA that codes for some of the mitochondrial proteins.

The overall process of oxidation of reduced coenzymes, reduction of oxygen to water, and phosphorylation of ADP to ATP requires intact mitochondria, or intact sealed vesicles of mitochondrial inner membrane prepared by disruption of mitochondria; it will not occur with solubilized preparations from mitochondria, or with open fragments of mitochondrial inner membrane. Under normal conditions, these three processes are linked, and none can occur without the others.

3.3.1.2 The mitochondrial electron transport chain

The mitochondrial electron transport chain is a series of enzymes and coenzymes in the crista membrane, each of which is reduced by the preceding coenzyme, and in turn reduces the next, until finally the protons and electrons that have entered the chain from either NADH or reduced flavin reduce oxygen to water. The sequence of the electron carriers shown in Figure 3.17 has been determined in two ways:

  • By consideration of their electrochemical redox potentials, which permits determination of which carrier is likely to reduce another, and which is likely to be reduced. There is a gradual fall in redox potential between the enzyme that oxidizes NADH and that which reduces oxygen to water.
  • By incubation of mitochondria with substrates, in the absence of oxygen, when all of the carriers become reduced, then introducing a limited amount of oxygen, and following the sequence in which the carriers become oxidized. The oxidation state of the carriers is determined by following changes in their absorption spectra.

Studies with inhibitors of specific electron carriers, and with artificial substrates that oxidize or reduce one specific carrier, permit dissection of the electron transport chain into four complexes of electron carriers:

Images Cytochrome Metabolism

cytochrome b cytochrome c-| cytochrome c

cytochrome oxidase (cytochromes a and a3)

Figure 3.17 An overview of the mitochondrial electron transport chain.

  • Complex I catalyses the oxidation of NADH and the reduction of ubiquinone, and is associated with the phosphorylation of ADP to ATP
  • Complex II catalyses the oxidation of reduced flavins and the reduction of ubiquinone. This complex is not associated with phosphorylation of ADP to ATP
  • Complex III catalyses the oxidation of reduced ubiquinone and the reduction of cytochrome c, and is associated with the phosphorylation of ADP to ATP;
  • Complex IV catalyses the oxidation of reduced cytochrome c and the reduction of oxygen to water, and is associated with the phosphorylation of ADP to ATP

In order to understand how the transfer of electrons through the electron transport chain can be linked to the phosphorylation of ADP to ATP, it is necessary to consider the chemistry of the various electron carriers. They can be classified into two groups:

• Hydrogen carriers, which undergo reduction and oxidation reactions involving both protons and electrons — these are NAD, flavins, and ubiquinone. As shown in Figure 2.17, NAD undergoes a two-electron oxidation/reduction reaction, while both the flavins (Figure 2.16) and ubiquinone (Figure 3.18) undergo two single-electron reactions to form a half-reduced radical, then the fully reduced coenzyme. Flavins can also undergo a two-electron reaction in a single step.

h3co u oxidized ubiqinone h3co

u oxidized ubiqinone

h3co.

h3co-

Color Ubiqinone

e"

Figure 3.18 Oxidation and reduction of ubiquinone (coenzyme Q).

Oxidation Reduction Coenzyme

II 10

half-reduced semi-quinone radical h3co.

e"

h3co-

fully reduced ubiquinol

Figure 3.18 Oxidation and reduction of ubiquinone (coenzyme Q).

  • Electron carriers, which contain iron (and in the case of cytochrome oxidase also copper); they undergo oxidation and reduction by electron transfer alone. These are the cytochromes, in which the iron is present in a haem molecule, and non-haem iron proteins, sometimes called iron—sulphur proteins, because the iron is bound to the protein through the sulphur of the amino acid cysteine. Figure 3.19 shows the arrangement of the iron in non-haem iron proteins and the three different types of haem that occur in cytochromes:
  • haem (protoporphyrin IX), which is tightly but non-covalently bound to proteins, including cytochromes b and bt, as well as enzymes such as catalase and the oxygen transport proteins haemoglobin and myoglobin;
  • haem C, which is covalently bound to protein in cytochromes c and c^
  • haem A, which is anchored in the membrane by its hydrophobic side-chain, in cytochromes a and a3 (which together form cytochrome oxidase).

The hydrogen and electron carriers of the electron transport chain are arranged in sequence in the crista membrane, as shown in Figure 3.20. Some carriers are entirely within the membrane, while others are located at the inner or outer face of the membrane.

There are two steps in which a hydrogen carrier reduces an electron carrier: the reaction between the flavin and non-haem iron protein in complex I and the reaction between ubiquinol and cytochrome b plus a non-haem iron protein in complex II.

haem (protoporphyrin IX)

haem (protoporphyrin IX)

=J CH2CH2COOH

CH2CH2COOH

=J CH2CH2COOH

CH2CH2COOH

CH2CH2COOH

CH2CH2COOH

CH2CH2COOH

CH2CH2COOH

CO ch3

CH2CH2COOH

CH2CH2COOH

OC CH NH OC CH NH

CH2 CH2

OC—CH—NH OC—CH—NH non-haem iron protein (iron sulphur protein)

haem C

Figure 3.19 Iron-containing carriers of the electron transport chain — haem and non-haem iron.

Electron Transport Chain
Figure 3.20 Complexes of the mitochondrial electron transport chain.

The reaction between non-haem iron protein and ubiquinone in complex I is the reverse — a hydrogen carrier is reduced by an electron carrier.

When a hydrogen carrier reduces an electron carrier, there is a proton that is not transferred onto the electron carrier but is extruded from the membrane, into the crista space, as shown in Figure 3.21.

When an electron carrier reduces a hydrogen carrier, there is a need for a proton to accompany the electron that is transferred. This is acquired from the mitochondrial matrix, thus shifting the equilibrium between H20 ^ H+ + OH-, resulting in an accumulation of hydroxyl ions in the matrix.

3.3.1.3 Phosphorylation of ADP linked to electron transport

The result of electron transport through the sequence of carriers shown in Figure 3.20, and the alternation between hydrogen carriers and electron carriers, is a separation of protons and hydroxyl ions across the crista membrane, with an accumulation of protons in the crista space and an accumulation of hydroxyl ions in the matrix — i.e. creation of a pH gradient across the inner membrane. This proton gradient provides the driving force for the phosphorylation of ADP to ATP, shown in Figure 3.22 — a highly endothermic reaction.

ATP synthase acts as a molecular motor, driven by the flow of protons down the concentration gradient from the crista space into the matrix, through the transmembrane stalk of the primary particle. As protons flow through the stalk, so they cause rotation of the core of the multienzyme complex that makes up the primary particle containing ATP synthase.

Electron Transport Chain Mitochondria
Figure 3.21 Hydrogen and electron carriers in the mitochondrial electron transport chain — generation of a transmembrane proton gradient.
Proton Gradient Mitochondria

OH OH

Figure 3.22 Condensation of ADP + phosphate ^ ATP.

OH OH

Figure 3.22 Condensation of ADP + phosphate ^ ATP.

Electron Transport Change

Figure 3.23 The mitochondrial ATP synthase — a molecular motor. As the central core rotates, so each site in turn undergoes a conformational change, A becoming equivalent to B, B to C and C to A.

Figure 3.23 The mitochondrial ATP synthase — a molecular motor. As the central core rotates, so each site in turn undergoes a conformational change, A becoming equivalent to B, B to C and C to A.

As shown in Figure 3.23, there are three ATP synthase catalytic sites in the primary particle, and each one-third turn of the central core causes a conformational change at each active site:

  • At one site the conformational change permits binding of ADP and phosphate.
  • At the next site the conformational change brings ADP and phosphate close enough together to undergo condensation and expel a proton and a hydroxyl ion.
  • At the third site the conformational change causes expulsion of ATP from the site, leaving it free to accept ADP and phosphate at the next part-turn.

At any time, one site is binding ADP and phosphate, one is undergoing condensation and the third is expelling ATP If ADP is not available to bind, then rotation cannot occur — and if rotation cannot occur, then protons cannot flow through the stalk from the crista space into the matrix.

3.3.1.4 The coupling of electron transport, oxidative phosphorylation and fuel oxidation

The processes of oxidation of reduced coenzymes and the phosphorylation of ADP to ATP are normally tightly coupled:

  • ADP phosphorylation cannot occur unless there is a proton gradient across the crista membrane resulting from the oxidation of NADH or reduced flavins.
  • If there is little or no ADP available, the oxidation of NADH and reduced flavins is inhibited, because the protons cannot cross the stalk of the primary particle, and so the proton gradient becomes large enough to inhibit further transport of protons into the crista space. Indeed, experimentally it is possible to force reverse electron transport and reduction of NAD+ and flavins by creating a proton gradient across the crista membrane.

Metabolic fuels can only be oxidized when NAD+ and oxidized flavoproteins are available. Therefore, if there is little or no ADP available in the mitochondria (i.e. it has all been phosphorylated to ATP), there will be an accumulation of reduced coenzymes, and hence a slowing of the rate of oxidation of metabolic fuels. This means that substrates are only oxidized when there is a need for the phosphorylation of ADP to ATP and ADP is available. The availability of ADP is dependent on the utilization of ATP in performing physical and chemical work, as shown in Figure 3.2.

It is possible to uncouple electron transport and ADP phosphorylation by adding a weak acid, such as dinitrophenol, that transports protons across the crista membrane. As shown in Figure 3.24, in the presence of such an uncoupler, the protons extruded during electron transport do not accumulate in the crista space but are transported into the mitochondrial matrix, where they react with hydroxyl ions, forming water. Under these conditions, ADP is not phosphorylated to ATP, and the oxidation of

Uncoupling Oxidative Phosphorylation
Figure 3.24 Uncoupling of electron transport and oxidative phosphorylation by a weak acid such as 2,4-dinitrophenol.

NADH and reduced flavins can continue unimpeded until all the available substrate or oxygen has been consumed. Figure 3.25 shows the oxygen electrode trace in the presence of an uncoupler.

The result of uncoupling electron transport from the phosphorylation of ADP is that a great deal of substrate is oxidized, with little production of ATP, although heat is produced. This is one of the physiological mechanisms for heat production to maintain body temperature without performing physical work — non-shivering thermogenesis. There are a number of proteins in the mitochondria of various tissues that act as proton transporters across the crista membrane when they are activated.

The first such uncoupling protein to be identified was in brown adipose tissue, and was called thermogenin because of its role in thermogenesis. Brown adipose tissue is anatomically and functionally distinct from the white adipose tissue that is the main site of fat storage in the body. It has a red—brown colour because it is especially rich in mitochondria. Brown adipose tissue is especially important in the maintenance of mitochondria + substrate added

Substrate Adp Dinitrophenol

time

Figure 3.25 Oxygen consumption by mitochondria incubated with malate and ADP, with and without an uncoupler.

time

Figure 3.25 Oxygen consumption by mitochondria incubated with malate and ADP, with and without an uncoupler.

body temperature in infants, but it remains active in adults, although its importance compared with uncoupling proteins in muscle and other tissues is unclear.

In addition to maintenance of body temperature, uncoupling proteins are important in overall energy balance and body weight (section 5.2). It was noted in section 1.3.2 that the hormone leptin secreted by (white) adipose tissue increases expression of uncoupling proteins in muscle and adipose tissue, so increasing energy expenditure and the utilization of adipose tissue fat reserves.

3.3.1.5 Respiratory poisons

Much of our knowledge of the processes involved in electron transport and oxidative phosphorylation has come from studies using inhibitors. Figure 3.26 shows the oxygen electrode traces from mitochondria incubated with malate and an inhibitor of electron transport, with or without the addition of dinitrophenol as an uncoupler. Inhibitors of electron transport include:

1 Rotenone, the active ingredient of derris powder, an insecticide prepared from the roots of the leguminous plant Lonchocarpus nicou. It is an inhibitor of complex I (NADH ^ ubiquinone reduction). The same effect is seen in the presence of amytal (amobarbital), a barbiturate sedative drug, which again inhibits complex I. These two compounds inhibit oxidation of malate, which requires complex I, but not succinate, which reduces ubiquinone directly. The addition of the uncoupler c o

Images Amobarbital Powder

time

Figure 3.26 Oxygen consumption by mitochondria incubated with malate and ADP, plus an inhibitor of electron transport, with and without an uncoupler.

time

Figure 3.26 Oxygen consumption by mitochondria incubated with malate and ADP, plus an inhibitor of electron transport, with and without an uncoupler.

has no effect on malate oxidation in the presence of these two inhibitors of electron transport, but leads to uncontrolled oxidation of succinate.

2 Antimycin A, an antibiotic produced by Streptomyces spp. that is used as a fungicide against fungi that are parasitic on rice. It inhibits complex III (ubiquinone ^ cytochrome c reduction). It inhibits the oxidation of both malate and succinate, as both require complex III, and the addition of the uncoupler has no effect.

3 Cyanide, azide or carbon monoxide, all of which bind irreversibly to the iron of cytochrome a3, and thus inhibit complex IV Again these compounds inhibit oxidation of malate and succinate, as both rely on cytochrome oxidase, and again the addition of the uncoupler has no effect.

Figure 3.27 shows the oxygen electrode traces from mitochondria incubated with malate and an inhibitor of ATP synthesis, with or without the addition of dinitrophenol as an uncoupler. Oligomycin is a therapeutically useless antibiotic produced by Streptomyces spp. that inhibits the transport of protons across the stalk of the primary particle. This results in inhibition of oxidation of both malate and succinate, because, if the protons cannot be transported back into the matrix, they will accumulate and mitochondria + substrate added

1000 nmol ADP ± inhibitor ± uncoupler added c o

Dintrophenol Inhibits Atp Synthesis

time

Figure 3.2 7 Oxygen consumption by mitochondria incubated with malate and ADP plus an inhibitor of ATP synthesis such as oligomycin, with and without an uncoupler.

time

Figure 3.2 7 Oxygen consumption by mitochondria incubated with malate and ADP plus an inhibitor of ATP synthesis such as oligomycin, with and without an uncoupler.

inhibit further electron transport. In this case, addition of the uncoupler permits reentry of protons across the crista membrane, and hence uncontrolled oxidation of substrates.

Two further compounds also inhibit ATP synthesis not by inhibiting the ATP synthase, but by inhibiting the transport of ADP into, and ATP out of, the mitochondria:

1 Atractyloside is a toxic glycoside from the rhizomes of the Mediterranean thistle Atractylis gummifera; it competes with ADP for binding to the carrier.

2 Bongkrekic acid is a toxic antibiotic formed by Pseudomonas cocovenenans growing on coconut; it is named after bongkrek, an Indonesian mould-fermented coconut product that becomes highly toxic when Pseudomonas outgrows the mould. It fixes the carrier protein at the inner face of the membrane, so that ATP cannot be transported out, nor ADP in.

Both compounds thus inhibit ATP synthesis, and therefore the oxidation of substrates. However, as with oligomycin (see Figure 3.27), addition of an uncoupler permits rapid and complete utilization of oxygen, as electron transport can now continue uncontrolled by the availability of ADP.

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Responses

  • consuelo de luca
    How does ADP facilitate oxygen consumption in coupled mitochondria?
    4 years ago
  • TAMZIN
    What catalyses production of atp synthesis of metabolic water?
    4 years ago

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