The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

A Challenge from Textbook Bioenergetics and Free Radical Chemistry
results do not challenge the chemiosmotic theory sensu 1961, only its later elaboration.
Conversely, verifications of the chemiosmotic theory have involved the way in which
mitochondria function, and in how this function can be inhibited or stimulated. They have
not, in particular, addressed directly the question of whether the immediate environment
of the mitochondrial membrane is (for these purposes) faithfully represented by
measurements of the medium some way away from the membrane.
11.3.3. Macroscopic Restriction of Ion Movement
One important factor that may well have caused Tedeschi’s work to be so poorly
received was that he never proposed an alternative hypothesis to Mitchell’s. In fairness,
though, it is indeed hard to see how an aqueous compartment can be anything other than
electrochemically homogeneous, because ions—especially protons—diffuse so fantastically
fast in water.
One candidate for a barrier to homogeneity 39 is the shape of the inner membrane. The
intricate cristae into which it is folded will inevitably hinder the flow of protons, and it is
reasonable to suppose that occasionally the membrane will come together and separate a
scrap of cytosol in the intermembrane space from the main bulk. Topologically, that scrap
of cytosol would then resemble the inside of a chloroplast’s thylakoid membrane: since it
was no longer in electrochemical contact with the cytosol, proton-pumping would be able
to acidify it sufficiently that the pH difference across the membrane could drive ATP synthesis
unaided. But this will probably not happen often enough, or anyway not for long enough at
a stretch, to affect the pH to a chemically significant degree. Moreover, we must recall that
some of Tedeschi’s experiments were performed with “giant” mitochondria, whose outer
membrane had been removed and whose inner membrane had then been swollen by osmosis
to its maximum possible size—in other words, ironing out all the cristae. And those
mitochondria still phosphorylated properly, with apparently not nearly as much bulk-tobulk
proton gradient as should be needed according to Mitchell. So this idea—which can be
termed the “pseudothylakoid hypothesis”—is not the answer.
11.3.4. Totally Localized Coupling
So far, we have seen that in order to reconcile Tedeschi’s results with Mitchell’s 1961
theory, protons must somehow be restrained from rapid movement in all three dimensions
within the cytosol and the matrix. There are two basic alternatives to three-dimensional
proton movement, and both have been explored: namely, two-dimensional and
one-dimensional. The one-dimensional version has been tested experimentally, and appears
not to be true. What it says is that the primary proton pumps (the enzymes of the respiratory
chain) and the secondary ones (the ATP synthases) are arranged in one-to-one juxtaposition,
either all the time or periodically, such that they pass protons between them by direct contact.
If this were so, protons would by and large never escape into the bulk aqueous phase, so we
would indeed not see a Δψ.
A rather elegant test of this involves noting that it predicts not only an inhibition of
proton movement into the bulk, but also inhibition of proton leak across the membrane. As
noted in Section 8.4, leak exists in an easily measurable amount; it varies with the
proton-motive force, as one would expect. Now, the proton gradient can be reduced by
adding an ionophore (a chemical that simply makes the membrane leakier to ions), such as
carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (mercifully abbreviated FCCP); and
it can also be reduced by adding ADP and phosphate, because that will stimulate the ATP
synthase to work faster and dissipate the proton gradient. So, if the ATP synthase receives its
protons only by a direct transfer from a respiratory chain enzyme, then adding ADP and
phosphate will cause a rapid loss of proton gradient and rise in respiration rate, whereas the
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