| Uncouplers are hydrophobic weak acids (Fig. 18-14). Their
hydrophobicity allows them to diffuse readily across mitochondrial membranes.
After entering the mitochondrial matrix in the protonated form, they can release
a proton (dissociate), thus dissipating the proton gradient. Ionophores uncouple
electron transfer from oxidative phosphorylation by creating electrical short
circuits across the mitochondrial membrane (Fig. 18-20). The toxic ionophore
valinomycin forms a lipid-soluble complex with K+,
which is abundant in the cytosol (see Fig. 10-26). Whereas
K+ penetrates the mitochondrial inner membrane only
very slowly, the K+-valinomycin complex readily passes
through. The influx of positive ions neutralizes the excess of negative charge
inside the matrix, diminishing the electrical component of the proton-motive
force. Valinomycin slows mitochondrial ATP synthesis without blocking electron
transfer to O2
If the role of electron transfer in mitochondrial ATP synthesis is
simply to pump protons to create the electrochemical potential of the
proton-motive force, an artificially created proton gradient should be able to
replace electron transfer in driving ATP synthesis. This prediction of the
chemiosmotic theory has been experimentally tested and confirmed (Fig. 18-21).
Mitochondria manipulated so as to impose a difference of proton concentration
and a separation of charge across the inner membrane (Fig. 18-21) carry out the
synthesis of ATP in the absence of an oxidizable substrate; the proton-motive
force alone suf fices to drive ATP synthesis.
Chemiosmotic theory also accounts for a third condition that
uncouples oxidation from phosphorylation-mechanical disruption of the
mitochondrial membrane. Without an intact membrane there can be no proton
gradient, hence no energy conservation and no ATP synthesis.
The Detailed Mechanism of ATP Formation ftemains Elusive
Although it is clear that a transmembrane proton gradient provides
the energy for ATP synthesis, it is not clear how this energy is transmitted to
the ATP synthase. This enzyme catalyzes the conversion of enzyme-bound ADP and
P i into bound ATP even in the absence of a proton gradient.
Remarkably, it appears that the reaction ADP + P i ![]() ATP + H 2O is readily
reversible-that the free-energy change for ATP synthesis on the enzyme surface
is close to zero. However, the ATP formed in this reaction remains tightly bound
to the active site, preventing further catalysis at that site. The proton-motive
force apparently supplies the energy needed to force dissociation of the tightly
bound ATP from the enzyme, allowing another catalytic cycle to begin.
How is it possible that ATP synthesis, known to be strongly
endergonic in aqueous solution (ΔG°' = 30.5 kJ/mol), is readily reversible on
the enzyme surface? The very tight noncovalent binding of enzyme to ATP may
supply enough binding energy (Chapter 8) to make bound ATP about as stable as
its hydrolysis products (Fig. 18-22). Alternatively, the reaction may occur in a
very hydrophobic pocket in the enzyme interior, where the energetics of
hydrolysis in water do not apply.
Each F1 complex has the subunit
composition α3β3γδε (Fig.
18-15). A tight binding site for ATP, apparently identical to the catalytic
site, is located on each β subunit, or perhaps between each β and its associated
a subunit. Every F1 complex therefore has three
ATP-synthesizing sites, which interact with F0 through
the single copies of γ, δ, and ε subunits.
On the basis of detailed kinetic and binding studies of the
reactions catalyzed by F0F1,
Paul Boyer has suggested a mechanism (Fig. 18-23) in which the three active
sites on F1 alternate in catalyzing ATP synthesis. The
limiting step in the process is the release of newly synthesized ATP from the
enzyme. A conformational transition driven by the proton-motive force reduces
the enzyme's affinity for ATP.
The transition induced by the proton-motive force may be envisioned as a 120°
rotation of the α3β3 portion
of F1 (Fig. 18-23), placing one of the three α-β pairs
in a special position relative to the proton channel of
F0. However, no physical rotation of
F1 has been demonstrated, and the model does not
require it; the conformational change that interconverts the three types of
sites may be an allosteric transition, in which changes at one α-β pair force
compensating changes in the other two pairs.
What makes the mechanism of this enzyme particularly difficult to solve is
the Uectorial nature of the process it catalyzes. Somehow, the enzyme must sense
not merely a certain concentration of protons, but a difference of proton
concentration in two regions of space. Determination of the detailed structure
of F1 by x-ray crystallography should shed light on
the mechanism of its action.
The Proton-Motive Force Energizes Active Transport
The primary role of electron transfer in mitochondria is to furnish energy
for the synthesis of ATP during oxidative phosphorylation, but this energy
serves also to drive several transport processes essential to oxidative
phosphorylation. We have seen that the inner mitochondrial membrane is generally
impermeable to charged species, but two specific systems in the inner
mitochondrial membrane transport ADP and Pi into the matrix and
ATP out to the cytosol (Fig. 18-24). The adenine nucleotide translocase, which
extends across the inner membrane, binds ADP3- on the
outside (cytosolic) surface of the inner membrane and transports it inward in
exchange for an ATP4- molecule simultaneously
transported outward (see Fig. 13-1 for the ionic forms of ATP and ADP). Because
this antiporter moves four negative charges out for each three moved in, its
activity is favored by the transmembrane electrochemical gradient, which gives
the matrix a net negative charge; the proton-motive force drives ATP-ADP
exchange.
Adenine nucleotide translocase is specifically inhibited by atractyloside, a
toxic glycoside formed by a species of thistle; for centuries it has been known
that grazing cattle are poisoned when they ingest this plant. If the transport
of ADP into and ATP out of the mitochondria is inhibited, cytosolic ATP cannot
be regenerated from ADP, explaining the toxicity of atractyloside
A second membrane transport system essential to oxidative phosphorylation is
the phosphate translocase, which promotes symport of one
H2P04-
and one H+ into the matrix. This transport process,
too, is favored by the transmembrane proton gradient (Fig. 18-24).
Shuttle Systems Are Required for Mitochondrial Oxidation of Cytosolic
NADH
The NADH dehydrogenase of the inner mitochondrial membrane of animal cells
can accept electrons only from NADH in the matrix. Given that the inner membrane
is not permeable to cytosolic NADH, how can the NADH generated by glycolysis
outside mitochondria be reoxidized to NAD+ by
O2 via the respiratory chain? Special shuttle systems
carry reducing equivalents from cytosolic NADH into mitochondria by an indirect
route. The most active NADH shuttle, which functions in liver, kidney, and heart
mitochondria, is the malate-aspartate shuttle (Fig. 18-25). The reducing
equivalents of cytosolic NADH are first transferred to cytosolic oxaloacetate to
yield malate by the action of cytosolic malate dehydrogenase. The malate thus
formed passes through the inner membrane into the matrix via the
malatea-ketoglutarate transport system. Within the matrix the reducing
equivalents are passed by the action of matrix malate dehydrogenase to matrix
NAD+, forming NADH; this NADH can then pass electrons
directly to the respiratory chain in the inner membrane. Three molecules of ATP
are generated as this pair of electrons passes to 02.
Cytosolic oxaloacetate must be regenerated via transamination reactions (see
Fig. 17-5) and the activity of membrane transporters (Fig. 18-25) to start
another cycle of the shuttle.
In skeletal muscle and brain, another type of NADH shuttle, the
glycerol-3-phosphate shuttle, occurs (Fig. 18-26). It differs from the
malate-aspartate shuttle in that it delivers the reducing equivalents from NADH
into Complex III, not Complex I (Fig. 18-9), providing only enough energy to
synthesize two ATP molecules per pair of electrons.
The mitochondria of higher plants have an externally oriented NADH
dehydrogenase that is able to transfer electrons directly from cytosolic NADH
into the respiratory chain.
The complete oxidation of a molecule of glucose to
CO2 yields two ATP and two NADH from glycolysis in the
cytosol (Chapter 14); two NADH from pyruvate oxidation in the mitochondrial
matrix and two ATP, six NADH, and two
FADH2 from citric acid cycle reactions in the matrix Each NADH produced in the matrix yields three ATP from
mitochondrial oxidative phosphorylation, and for each
FADH2, two ATP are generated. Cytosolic NADH, after
shuttling into the matrix, yields two or three ATP, depending on which shuttle
is used. The total yield of glucose oxidation is therefore 36 or 38 ATP per
glucose
By comparison, glycolysis under anaerobic conditions (lactate fermentation)
yields only two ATP per glucose. Clearly, the evolution of oxidative
phosphorylation provided a tremendous increase in the energetic efficiency of
catabolism.
The oxidation of fatty acids and amino acids also takes place within the
mitochondrial matrix, and the FADH2 and NADH produced
by these oxidative pathways also serve as electron donors for oxidative
phosphorylation. Complete oxidation of the 16-carbon saturated fatty acid
palmitate to CO2 produces eight acetyl-CoAs, seven
FADH2, and seven NADH. The oxidation of
each acetyl-CoA via the citric acid cycle produces three NADH, one
FADH2, and one ATP (GTP). The gain in ATP is therefore
131 ATP but, because the activation of palmitate to palmitoyl-CoA
costs two ATP equivalents, the net gain is 129 ATP per palmitate. A similar
calculation may be made for the ATP yield upon oxidation of each of the amino
acids. The important conclusion here is that aerobic oxidative
pathways that result in electron transfer to O2
accompanied by oxidative phosphorylation account for the vast majority of the
ATP produced in catabolism.
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