The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

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The Mitochondrial Free Radical Theory of Aging
postulates he had originally discussed (and all of which had, even by 1966, already been
quite thoroughly confirmed by experiment). This new assertion is that the bulk aqueous
phases inside and outside the mitochondrial inner membrane are each electrochemically
homogeneous: that the combination of the electrical potential and the pH is the same
everywhere in the matrix and also the same everywhere in the cytosol, so that the difference
between the two—the proton-motive force, as Mitchell had defined it—would be the same
when measured between any point in the cytosol and any point in the matrix as it was
across any of the OXPHOS enzymes. This model is known as the delocalized chemiosmotic
theory.
This may seem, at first hearing, to be an altogether uncontroversial assertion. After all,
the pH and electrical potential are formed by protons and other ions, all of which can surely
move freely throughout the aqueous space that they occupy (the matrix or the cytosol), so it
seems hard to question the assertion, i.e. to propose a proton-motive force between two
points within the same aqueous compartment. Indeed, no one really did question it—until
they were forced to.
Researchers of course sought to measure the proton-motive force between the cytosol
and the matrix in order to test this refined chemiosmotic hypothesis. This was not very easy,
though, because mitochondria are so small. One cannot get an electrode inside a mitochondrion,
for example. Therefore, measurements were made by indirect means: by measuring
the rate at which detectable (usually fluorescent) ions passed through the membrane in
appropriate conditions, and calculating how strongly they were being pushed. 33,34 These
experiments confirmed that the proton-motive force was about what it needed to be to
drive ATP synthesis.
But in 1969, Henry Tedeschi and colleagues reported 35 a much lower—indeed,
negligible—value. This would have been unexciting if their method of measuring it had
been similar to what others had used; it is, after all, not uncommon in science for experiments
whose design initially seems valid to be found at fault in later years. But they had used a
method which was not the same at all, and which, most importantly, was far more direct.
They had succeeded in doing what I just said was impossible—getting an electrode inside a
mitochondrion, thus allowing them to measure the potential difference between the matrix
and the cytosol purely electrically, avoiding any inferences based on the behaviour of
chemicals not present in vivo. They did this initially by using mitochondria that are a great
deal larger than normal, and in later experiments by making normal mitochondria swell.
This result was not well received. For the next decade and more, bioenergeticists raised
challenge after challenge to the validity of Tedeschi’s techniques and/or results. Each time,
he and his coworkers responded by improving the experimental design so as to confirm that
the result was real. In the late 1970s, they succeeded in showing that the mitochondria were
generating ATP at the usual rate, even while they were impaled by an electrode and their Δψ
was being measured (and found to be about zero). 36 They also showed, by ingenious use of
a mitochondrion impaled by two electrodes, that the impalement was real—that the electrode
was not just encased in an invagination of the (unpunctured) membrane. 37 Finally, in 1984
they eliminated the possibility that the swelling of the mitochondrion had somehow lowered
its internal pH, allowing ΔpH to drive ATP synthesis unaided. 38
Most specialists, however, remain sure to this day that, robust though the evidence
appears to be that these measurements are reliable, the chemiosmotic theory is simply too
well confirmed to be rejected on this basis. (No discussion whatever has appeared regarding
the challenge to Mitchell’s model posed by the superoxide dismutation results discussed in
Section 11.2.3, 21 doubtless because they were not presented as such.) They have decided
that there must be something wrong with the experiments that report inadequate Δψ, even
though exactly what is wrong has not been established. But, as stressed above, Tedeschi’s