Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

Independence of the Four Carrier Complexes

It should be emphasized here that the four major complexes of the electron transport chain operate quite independently in the inner mitochondrial membrane. Each is a multiprotein aggregate maintained by numerous strong associations between peptides of the complex, but there is no evidence that the complexes associate with one another in the membrane. Measurements of the lateral diffusion rates of the four complexes, of coenzyme Q, and of cytochrome c in the inner mitochondrial membrane show that the rates differ considerably, indicating that these complexes do not move together in the membrane. Kinetic studies with reconstituted systems show that electron transport does not operate by means of connected sets of the four complexes.

A Dynamic Model of Electron Transport

The model that emerges for electron transport is shown in Figure 21.21. The four complexes are independently mobile in the membrane. Coenzyme Q collects electrons from NADH–UQ reductase and succinate–UQ reductase and delivers them (by diffusion through the membrane core) to UQ–cyt c reductase. Cytochrome c is water-soluble and moves freely, carrying electrons from UQ–cyt c reductase to cytochrome c oxidase. In the process of these electron transfers, protons are driven across the inner membrane (from the matrix side to the intermembrane space). The proton gradient generated by electron transport represents an enormous source of potential energy. As seen in the next section, this potential energy is used to synthesize ATP as protons flow back into the matrix.

The H /2e Ratio for Electron Transport Is Uncertain

In 1961, Peter Mitchell, a British biochemist, proposed that the energy stored in a proton gradient across the inner mitochondrial membrane by electron transport drives the synthesis of ATP in cells. The proposal became known as

R2 R1

CuB2+

Heme

FIGURE 21.19 ● The binuclear center of cytochrome oxidase. A ligand, L (probably a cysteine S), is shown bridging the CuB and Fea3 metal sites.

● A model for the mechanism of O2 reduction by cytochrome oxidase.

(Adapted from Nicholls, D. G., and Ferguson, S. J., 1992. Bioenergetics 2. London: Academic Press; and Babcock, G. T., and Wikström, M., 1992. Nature 356:301–309.)

4 +

Intermembrane space

I

Matrix

4 H+

FIGURE 21.21 ● A model for the electron transport pathway in the mitochondrial inner membrane. UQ/UQH2 and cytochrome c are mobile electron carriers and function by transferring electrons between the complexes. The proton transport driven by Complexes I, III, and IV is indicated.

H2O

Mitchell’s chemiosmotic hypothesis. The ratio of protons transported per pair of electrons passed through the chain—the so-called H /2 e ratio—has been an object of great interest for many years. Nevertheless, the ratio has remained extremely difficult to determine. The consensus estimate for the electron transport pathway from succinate to O2 is 6 H /2 e . The ratio for Complex I by itself remains uncertain, but recent best estimates place it as high as 4 H /2 e . On the basis of this value, the stoichiometry of transport for the pathway from NADH to O2 is 10 H /2 e . Although this is the value assumed in Figure 21.21, it is important to realize that this represents a consensus drawn from many experiments.

For many years, the means by which electron transport and ATP synthesis are coupled was unknown. It is no exaggeration to say that the search for the coupling mechanism was one of the largest, longest, most bitter fights in the history of biochemical research. Since 1777, when the French chemist Lavoisier determined that foods undergo oxidative combustion in the body, chemists and biochemists have wondered how energy from food is captured by living things. A piece of the puzzle fell into place in 1929, when Fiske and Subbarow first discovered and studied adenosine 5 -triphos- phate in muscle extracts. Soon it was understood that ATP hydrolysis provides the energy for muscle contraction and other processes.

Engelhardt’s experiments in 1930 led to the notion that ATP is synthesized as the result of electron transport, and, by 1940, Severo Ochoa had carried out a measurement of the P/O ratio, the number of molecules of ATP generated per atom of oxygen consumed in the electron transport chain. Because two electrons are transferred down the chain per oxygen atom reduced, the P/O ratio also reflects the ratio of ATPs synthesized per pair of electrons consumed. After many tedious and careful measurements, scientists decided that the P/O ratio was 3 for NADH oxidation and 2 for succinate (that is, [FADH2]) oxidation. Electron flow and ATP synthesis are very tightly coupled in the sense that, in normal mitochondria, neither occurs without the other.

A High-Energy Chemical Intermediate Coupling

Oxidation and Phosphorylation Proved Elusive

Many models were proposed to account for the coupling of electron transport and ATP synthesis. A persuasive model, advanced by E. C. Slater in 1953, proposed that energy derived from electron transport was stored in a high-energy intermediate (symbolized as X P). This chemical species—in essence an activated form of phosphate—functioned according to certain relations according to Equations (21.22)–(21.25) (see below) to drive ATP synthesis.

This hypothesis was based on the model of substrate-level phosphorylation in which a high-energy substrate intermediate is a precursor to ATP. A good example is the 3-phosphoglycerate kinase reaction of glycolysis, where 1,3-bisphosphoglycerate serves as a high-energy intermediate leading to ATP. Literally hundreds of attempts were made to isolate the high-energy intermediate, X P. Among the scientists involved in the research, rumors that one group or another had isolated X P circulated frequently, but none was substantiated. Eventually it became clear that the intermediate could not be isolated because it did not exist.

Peter Mitchell’s Chemiosmotic Hypothesis

In 1961, Peter Mitchell proposed a novel coupling mechanism involving a proton gradient across the inner mitochondrial membrane. In Mitchell’s chemiosmotic hypothesis, protons are driven across the membrane from the matrix to the intermembrane

space and cytosol by the events of electron transport. This mechanism stores the energy of electron transport in an electrochemical potential. As protons are driven out of the matrix, the pH rises and the matrix becomes negatively charged with respect to the cytosol (Figure 21.22). Proton pumping thus creates a pH gradient and an electrical gradient across the inner membrane, both of which tend to attract protons back into the matrix from the cytoplasm. Flow of protons down this electrochemical gradient, an energetically favorable process, then drives the synthesis of ATP.

Paul Boyer and the Conformational Coupling Model

Another popular model invoked what became known as conformational coupling. If the energy of electron transport was not stored in some high-energy intermediate, perhaps it was stored in a high-energy protein conformation. Proposed by Paul Boyer, this model suggested that reversible conformation changes transferred energy from proteins of the electron transport chain to the enzymes involved in ATP synthesis. This model was consistent with some of the observations made by others, and it eventually evolved into the binding change mechanism (the basis for the model in Figure 21.28). Boyer’s model is supported by a variety of binding experiments and is essentially consistent with Mitchell’s chemiosmotic hypothesis.

Electron transport drives H+ out and creates an electrochemical gradient

lower [H+] in matrix

Lower pH,

higher [H+] in intermembrane space

FIGURE 21.22 ● The proton and electrochemical gradients existing across the inner mitochondrial membrane. The electrochemical gradient is generated by the transport of protons across the membrane.

● Electron micrograph of submitochondrial particles showing the 8.5-nm projections or particles on the inner membrane, eventually shown to be F1–ATP synthase.

(Parsons, D. F., 1963. Science 140:985)

which is the free energy change for movement of a mole of protons across a typical inner membrane. Note that the free energy terms for both the pH difference and the potential difference are unfavorable for the outward transport of protons, with the latter term making the greater contribution. On the other hand, the G for inward flow of protons is 23.3 kJ/mol. It is this energy that drives the synthesis of ATP, in accord with Mitchell’s model. Peter Mitchell was awarded the Nobel Prize in chemistry in 1978.

21.9 ● ATP Synthase

The mitochondrial complex that carries out ATP synthesis is called ATP synthase or sometimes F1F0–ATPase (for the reverse reaction it catalyzes). ATP synthase was observed in early electron micrographs of submitochondrial particles (prepared by sonication of inner membrane preparations) as round, 8.5- nm-diameter projections or particles on the inner membrane (Figure 21.23). In micrographs of native mitochondria, the projections appear on the matrixfacing surface of the inner membrane. Mild agitation removes the particles from isolated membrane preparations, and the isolated spherical particles catalyze ATP hydrolysis, the reverse reaction of the ATP synthase. Stripped of these particles, the membranes can still carry out electron transfer but cannot synthesize ATP. In one of the first reconstitution experiments with membrane proteins, Efraim Racker showed that adding the particles back to stripped membranes restored electron transfer–dependent ATP synthesis.

ATP Synthase Consists of Two Complexes—F1 and F0

ATP synthase actually consists of two principal complexes. The spheres observed in electron micrographs make up the F1 unit, which catalyzes ATP synthesis. These F1 spheres are attached to an integral membrane protein aggregate called the F0 unit. F1 consists of five polypeptide chains named , , , , and , with a subunit stoichiometry 3 3 (Table 21.3). F0 consists of three hydrophobic subunits denoted by a, b, and c, with an apparent stoichiometry of a1b2c9–12. F0 forms the transmembrane pore or channel through which protons move to drive ATP synthesis. The , , , , and subunits of F1 contain 510, 482, 272, 146, and 50 amino acids, respectively, with a total molecular mass

Table 21.3

Escherichia coli F1F0 ATP Synthase Subunit Organization

for F1 of 371 kD. The and subunits are homologous, and each of these subunits bind a single ATP. The catalytic sites are in the subunits; the function of the ATP sites in the subunits is unknown (deletion of the sites does not affect activity).

John Walker and his colleagues have determined the structure of the F1 complex (Figure 21.24). The F1–ATPase is an inherently asymmetrical structure, with the three subunits having three different conformations. In the structure solved by Walker, one of the -subunit ATP sites contains AMP-PNP (a nonhydrolyzable analog of ATP), and another contains ADP, with the third site being empty. This state is consistent with the binding change mechanism for ATP synthesis proposed by Paul Boyer, in which three reaction sites cycle concertedly through the three intermediate states of ATP synthesis (take a look at Figure 21.28 on page 697).

How might such cycling occur? Important clues have emerged from several experiments that show that the subunit rotates with respect to the complex. How such rotation might be linked to transmembrane proton flow and ATP synthesis is shown in Figure 21.25. In this model, the c subunits of F0

(b)

δ

H+

F1

b

ε

γ

Fo

(a)

(b)

FIGURE 21.24 ● Molecular graphic images

(a) side view and (b) top view of the F1–ATP synthase showing the individual component peptides. The -subunit is the pink structure visible in the center of view (b).

ADP + P

ADP +

P

ATP

Energy

ADP +

P

ATP

ATP

H2O

ATP

T

L O

ADP + P

Cycle repeats

...

H

O

CH3O

OCH3

Rotenone

O O

S C C

CF3

2-Thenoyltrifluoroacetone

Carboxin

Carbonyl cyanide-p-trifluoro- methoxyphenyl hydrazone

—best known as FCCP; for Fluoro Carbonyl Cyanide Phenylhydrazone

Another important class of reagents affects ATP synthesis, but in a manner that does not involve direct binding to any of the proteins of the electron transport chain or the F1F0–ATPase. These agents are known as uncouplers because they disrupt the tight coupling between electron transport and the ATP synthase. Uncouplers act by dissipating the proton gradient across the inner mitochondrial membrane created by the electron transport system. Typical examples include 2,4-dinitrophenol, dicumarol, and carbonyl cyanide-p-trifluoro- methoxyphenyl hydrazone (perhaps better known as fluorocarbonyl-cyanide phenylhydrazone or FCCP) (Figure 21.31). These compounds share two common features: hydrophobic character and a dissociable proton. As uncouplers, they function by carrying protons across the inner membrane. Their tendency is to acquire protons on the cytosolic surface of the membrane (where the proton concentration is high) and carry them to the matrix side, thereby destroying the proton gradient that couples electron transport and the ATP synthase. In mitochondria treated with uncouplers, electron transport continues, and protons are driven out through the inner membrane. However, they leak back in so rapidly via the uncouplers that ATP synthesis does not occur. Instead, the energy released in electron transport is dissipated as heat.

Endogenous Uncouplers Enable Organisms To Generate Heat

Ironically, certain cold-adapted animals, hibernating animals, and newborn animals generate large amounts of heat by uncoupling oxidative phosphorylation. Adipose tissue in these organisms contains so many mitochondria that it is called brown adipose tissue for the color imparted by the mitochondria. The inner membrane of brown adipose tissue mitochondria contains an endogenous protein called thermogenin (literally, “heat maker”), or uncoupling protein, that creates a passive proton channel through which protons flow from the cytosol to the matrix. Certain plants also use the heat of uncoupled proton transport for a special purpose. Skunk cabbage and related plants contain floral spikes that are maintained as much as 20 degrees above ambient temperature in this way. The warmth of the spikes serves to vaporize odiferous molecules, which attract insects that fertilize the flowers.

● Structures of several uncouplers, molecules that dissipate the proton gradient across the inner mitochondrial membrane and thereby destroy the tight coupling between electron transport and the ATP synthase reaction.