We now turn to the most fundamental question about mitochondrial oxidative
phosphorylation: how does the flow of electrons through the respiratory chain
channel energy into the synthesis of ATP? We have seen that electron transfer
through the respiratory chain releases more than enough free energy to form ATP.
Mitochondrial oxidative phosphorylation therefore poses no thermodynamic
problem. However, one cannot deduce from thermodynamic considerations the
chemical mechanism by which energy released in one exergonic reaction (the
oxidation of NADH by O
2) is channeled into a second,
endergonic, reaction (the condensation of ADP and Pi). To describe the process
of oxidative phosphorylation completely, we need to identify the physical and
chemical changes that result from electron flow and cause ADP phosphorylation -
the mechanism that couples oxidation with phosphorylation.
We begin our discussion by considering the stoichiometry of oxidation and
phosphorylation in isolated mitochondria and the evidence for obligatory
coupling of the two processes. The chemiosmotic interpretation of oxidative
phosphorylation is then presented, with the major lines of evidence that support
it. The enzyme ATP synthase, which is directly responsible for ATP synthesis, is
the equivalent of an F-type ATP-dependent proton pump working in reverse; the
flow of protons down their electrochemical gradient through this "pump" drives
the condensation of P
i and ADP. We describe also the membrane
transport systems that move substrates, products, and reducing equivalents
between the cytosol and the mitochondrial matrix. Having looked in detail at the
coupling of ATP synthesis to electron flow, we will see that in the mitochondria
of some tissues the two processes are deliberately "uncoupled" to produce
heat.
We conclude with a summary of the overall regulation of ATPproducing
processes in the cell, and a look at two further interesting aspects of
mitochondria: the mitochondrial genome (and the effects of mutations therein)
and the likely evolutionary origins of these organelles.
Phosphorylation of ADP Is Coupled to Electron Transfer
When isolated mitochondria are suspended in a buffer containing ADP,
P
i, and an oxidizable substrate such as succinate, three easily
measured processes occur: (1) the substrate is oxidized (succinate yields
fumarate), (2) O
2 is consumed (respiration occurs),
and (3) ATP is synthesized. Careful experimental measurements of the
stoichiometry of electron transfer to O
2 and the
associated synthesis of ATP show that with NADH as electron donor, mitochondria
synthesize nearly 3.0 ATP per pair of electrons passed to
O
2, and with succinate nearly 2.0 ATP per electron
pair. Oxygen consumption and ATP synthesis are dependent upon substrate
oxidation, as can be seen in the experiments diagrammed in Figure 18-13.
Because the energy of substrate oxidation drives ATP synthesis in
mitochondria, it is not surprising that inhibitors of the passage of electrons
to O
2 (e.g., cyanide ion, carbon monoxide, and
antimycin A) block ATP synthesis (Fig. 18-13a). It is perhaps not so obvious
that the converse is true: inhibition of ATP synthesis blocks electron transfer
in intact mitochondria. This obligatory coupling can be demonstrated in isolated
mitochondria by providing O
2 and oxidizable
substrates, but not ADP (Fig. 18-13b). Under these conditions, no ATP synthesis
can occur, and electron transfer to O
2 is also
strikingly reduced. Coupling of oxidation and phosphorylation can also be
demonstrated using oligomycin or venturicidin, toxic antibiotics that bind to
the ATP synthase in mitochondria. These compounds are potent inhibitors of both
ATP synthesis and the transfer of electrons through the chain of carriers to
O
2 (Fig. 18-13b). Because oligomycin is known not to
interact directly with the electron carriers but only with ATP synthase, it
follows that electron transfer and ATP synthesis are obligatorily coupled;
neither reaction occurs without the other.
There are, however, certain conditions and reagents that uncouple
oxidation from phosphorylation. When intact mitochondria are disrupted by
treatment with detergent or physical shear, the resulting memfbrane fragments
are still capable of catalyzing electron transfer from succinate or NADH to
O2, but no ATP synthesis is coupled to this
respiration. Certain chemical compounds also cause uncoupling (Fig. 18-13b),
without disrupting mitochondrial structure. The chemical uncouplers (Table 18-4)
include 2,4-dinitrophenol (DNP) and a group of compounds related to
carbonylcyanide phenylhydrazone (Fig. 18-14). All of these uncouplers are weak
acids with hydrophobic properties. Ionophores (p. 560) also uncouple oxidative
phosphorylation. These agents bind to inorganic ions and surround them with
hydrophobic moieties; the ionophore-metal ion complexes pass easily through
membranes. We shall see later how the chemiosmotic theory accounts for the
action of uncouplers.
ATP Synthase Is a Large Membrane-Protein Complex
ATP synthase, the ATP-synthesizing enzyme complex of the inner mitochondrial
membrane, has two major components (or factors), Fl and Fo (Fig. 18-15). The
subscript letter 0 in F0 denotes that it is the
portion of the ATP synthase that confers sensitivity to oligomycin, a potent
inhibitor of this enzyme complex and thus of oxidative phosphorylation
F1 was first extracted from the mitochondrial inner
membrane and purified by Efraim Racker and his colleagues in the early 1960s.
Isolated F1 cannot synthesize ATP from ADP and
Pi; because it can catalyze the reverse reaction-hydrolysis of
ATP-the enzyme was originally called F1ATPase. When
F1 is carefully extracted from inside-out vesicles
prepared from the inner mitochondrial membrane (Fig. 18-16, p. 558), the
vesicles still contain intact respiratory chains and can catalyze electron
transfer, but cannot make ATP. When a preparation of isolated
F1 is added back to such depleted vesicles, their
capacity to couple electron transfer and ATP synthesis is restored. Membrane
reconstitution experiments of this kind, pioneered by Racker, opened new doors
to research on membrane structure and function.
F1, which in all aerobic organisms
consists of six subunits, contains several binding sites for ATP and ADP,
including the catalytic site for ATP synthesis. It is a peripheral membrane
protein complex, held to the membrane by its interaction with Fo, an integral
membrane protein complex of four different polypeptides that forms a
transmembrane channel through which protons can cross the membrane.
Highresolution electron micrographs of the isolated FoFI complex show the
knoblike F1 head, a stalk, and a base piece (Fo),
which normally extends across the inner membrane (Fig. 18-15b).
The complete F0F1
complex, like isolated F1, can hydrolyze ATP to ADP
and Pi, but its biological function is to catalyze the
condensation of ADP and Pi to form ATP. The
F0F1 complex is therefore
more appropriately called ATP synthase.
ATP Synthase Is Related to ATP-Dependent Proton Pumps
The structure and catalytic activity of ATP synthase from mitochondria show
that it belongs to a larger class of enzymes that we have already encountered:
the F-type ATPases (see Table 10-5). These protein complexes share the FoFI
structure, and there is sequence homology between the subunits of the F-type
ATPases and those of mitochondrial ATP synthase. F-type ATPases are
ATP-dependent proton pumps; they use the energy released by ATP hydrolysis to
move protons across membranes against a concentration gradient, thereby creating
a difference of pH and electrical charge (an electrochemical potential) across
the membrane. The F-type ATPases must have evolved very early; they are found in
the membranes of eubacteria and archaebacteria and in the mitochondria of all
aerobic eukaryotes. Chloroplasts also contain an ATP synthase that is an F-type
ATPase, which acts in light-driven ATP synthesis, as we shall see shortly.
The Chemiosmotic Model: A Proton Gradient couples Electron Flow and
Phosphorylation
How does the oxidation of substrates via electron transfer through the
respiratory chain cooperate with the ATP synthase to bring about phosphorylation
of ADP to ATP? Early investigators of mitochondrial oxidative phosphorylation
had before them a well-documented example of an oxidation coupled with a
phosphorylation and involving a high-energy chemical intermediate: the
glyceraldehyde-3-phosphate dehydrogenase reaction. In this glycolytic step,
glyceraldehyde-3-phosphate is oxidized and simultaneously converted to
1,3-bisphosphoglycerate, a compound with a high-energy group at the site of the
oxidation (see Fig. 14-5). ATP is formed when 1,3-bisphosphoglycerate transfers
its activated Pi to ADP. The formal similarity between this
oxidative phosphorylation and that which takes place in mitochondria led
naturally to the chemical coupling hypothesis, according to which some
high-energy chemical intermediate is the direct product of electron transfer
through one of three coupling sites along the chain of mitochondrial electron
carriers. The energy in this putative chemical intermediate would then be used
to drive the synthesis of ATP. An enormous amount of effort was expended in the
search for such a chemical intermediate; none was found.
In the early 1960s Peter Mitchell suggested a new paradigm that has become
central to current thinking and research on biological energy transductions.
Mitchell's chemiosmotic hypothesis accounted for the experimental observations
and made certain novel and experimentally testable predictions. Having survived
more than two decades of vigorous testing and refinement, the hypothesis has
become a generally accepted theory that is applied not only to mitochondrial
oxidative phosphorylation, but to a wide array of other energy transductions,
including light-driven ATP formation in photosynthetic organisms.
As postulated in the chemiosmotic theory (Fig. 18-17), the
transfer of electrons along the respiratory chain is accompanied by outward
pumping of protons across the inner mitochondrial membrane, which results in a
transmembrane difference in proton concentration (a proton gradient) and thus in
pH; the matrix becomes alkaline relative to the cytosolic side of the membrane.
The electrochemical energy inherent in this difference in proton concentration
and separation of charge, the proton-motive force, represents a conservation of
part of the energy of oxidation. The proton-motive force is
subsequently used to drive the synthesis of ATP catalyzed by Fl as protons flow
passively back into the matrix through proton pores formed by
F0. To emphasize this crucial role of the
proton-motive force, the equation for ATP synthesis is sometimes written
In the more general case, the electrochemical energy of a
transmembrane gradient of any charged species is seen to be interconvertible
with the energy of chemical bonds. The energy stored in such a gradient has two
components, as implied in the word "electrochemical." One is the chemical
potential energy due to the difference in concentration of a chemical species in
the two regions separated by the membrane; the other is the electrical potential
energy that results from the separation of charge when an ion moves across the
membrane without its counterion
For the pumping of ions against an electrochemical gradient, both terms on
the right-hand side of this equation have positive values, and ΔG is therefore
positive. When protons flow spontaneously down their electrochemical gradient,
an amount of free energy equal to ΔG becomes available to do work; it is the
proton-motive force.
Chemiosmotic theory readily explains the dependence of electron transfer on
ATP synthesis in mitochondria (Fig. 18-17). When ATP synthesis is blocked (with
oligomycin, for example), protons cannot flow into the matrix through the
F0F1 complex. With no path
for the return of protons to the matrix and the continued extrusion of protons
driven by the activity of the respiratory chain, a large proton gradient, hence
a large proton-motive force, builds up. When the cost (free energy) of pumping
one more proton out of the matrix against this gradient equals or exceeds the
energy released by the transfer of electrons from NADH to
O2, electron flow must stop; the free energy for the
overall process of electron flow coupled to proton pumping becomes zero, and
equilibrium is attained. |
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