The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki
The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki
142 The Mitochondrial Free Radical Theory of Aging the aqueous medium, and for some phospholipids this group is acidic. Being acidic means that each head group easily loses a proton, leaving it negatively charged so that it attracts free protons into its vicinity. The pH in the water right next to a typical phospholipid membrane, therefore, is considerably lower than in the bulk medium further away from the membrane 14 (Fig. 11.2). The inner mitochondrial membrane has about 20% acidic phospholipids, which gives a pH at the surface about one unit* lower than in the bulk, 17a so there will indeed be plenty of HO2• there. Being neutral, HO2• can also pass through the membrane into the matrix; this may be an important mechanism of proton leak (see Section 8.4), 17b though unlike straightforward “water wire” transfer of protons it may not be promoted by lipid peroxidation. 11.2.3. Perhydroxyl and SOS Now for the relevance to SOS. It comes from the fact that pH is, quite simply, concentration of protons in the aqueous medium, and protons are what the respiratory chain pumps. A mtDNA mutation that inhibits the respiratory chain will, therefore, lower the proton concentration—that is, raise the pH—at the outer surface of its host mitochondrion’s inner membrane. This will reduce the protonation of superoxide to HO2• and consequently lower the rate of lipid peroxidation.** It has been argued 18 that all superoxide production must be on the matrix side of the inner membrane, but the evidence cited in support of this 9,19 actually only tells us that some is made inside. Moreover, there is experimental support for this effect of respiration rate on HO2• levels. Experiments in 1995 in the laboratory of Joe McCord, the codiscoverer of superoxide dismutase, investigated the effect of respiration on non-enzymatic dismutation of superoxide. Recall from Table 3.3 that non-enzymatic superoxide dismutation does not occur as a reaction between two superoxide anions, but instead either between two perhydroxyls or between one superoxide and one perhydroxyl. (The two-superoxide reaction is not totally absent, in fact, but it is at least six orders of magnitude slower than the others). 20 Therefore, the rate of non-enzymatic dismutation is a measure of pH. And indeed, McCord’s laboratory established that non-enzymatic dismutation of externally generated O2• — in the presence of isolated mitochondria was much faster when their respiration was rapid than when it was inhibited. 21 On top of all this, a perusal of Table 3.3 shows us that even the levels of HO• may fall when respiration slows! This is worth explaining, because the simulation noted above 12 is currently the only work showing that HO2• initiates most lipid peroxidation, and many biologists (with history on their side, I fully acknowledge) are very wary of simulations. The reason is that reaction 8 in Table 3.3, which is essentially the only way that HO• is made in vivo, requires not only superoxide but also H2O2, which is created by superoxide dismutation. Now, the intermembrane space is one place in the cell where superoxide may be plentiful but superoxide dismutase is absent,*** because the cytosolic version of the enzyme is too * The literature is strewn with statements of this pH difference, varying from 2.5 15 to only 0.3; 16 but these have discussed either unrealistic membrane composition or the pH not quite at the surface. The calculation cited here 17a is based firmly on well-supported measurements of the relevant biological parameters. ** The same argument implies that loss of respiratory capacity will lower the pH at the inner surface of the inner membrane, so increasing the levels of HO2• there. This increase will be much less, however, than the decrease in HO2• levels at the outer surface, since the pH inside will always remain above the value of about 6.5 generated by the head groups. Thus the net HO2• concentration on either side of the membrane will be reduced by an inhibition of respiration. *** SOD was originally reported 22 to be present in the intermembrane space, but more recent studies 23,24a have refuted this. The existence of a compartment lacking SOD may explain why birds have paradoxically low antioxidant enzyme levels (see Section 6.5.6). 24b
A Challenge from Textbook Bioenergetics and Free Radical Chemistry Fig. 11.2. Variation of pH near a phospholipid membrane. big (as are nearly all proteins) to fit through the pores in the outer membrane and the mitochondrial version is similarly trapped by the inner membrane. Therefore, the H2O2 formed in the intermembrane space is that formed by non-enzymatic dismutation, which (as noted above) slows down when respiration slows down. This has been further confirmed by the histochemical detection of singlet oxygen in the intermembrane space; 24c recall from Table 3.3 that singlet oxygen is generated by the non-enzymatic, but not the enzymatic, dismutation reaction. 24d Most analyzes have ignored non-enzymatic dismutation and therefore presumed that the H2O2 in the intermembrane space gets there by diffusion from either the cytosol or the matrix (where it was formed by superoxide dismutase); but if that is wrong, then the overall level of H2O2 may well fall when respiration slows, even if the level of superoxide rises. Yet another reason why this is so derives from the presence in the intermembrane space of cytochrome c, which (when in the ferric state) assiduously detoxifies superoxide back to oxygen by accepting its lonely electron, 25 but which also (when in the ferrous state) just as readily 26 donates an electron to HO2•, forming—yes—H2O2. This too would, therefore, happen at a slower rate if there were less HO2• present. Again it may seem as though I have comprehensively rebutted the challenge with which this chapter began. Again, the truth is very different. 11.3. Mitchell’s Oversimplification I may as well warn the reader honestly, in advance, that this section is the most arcane in the book. Bioenergetics is a challenging discipline at the best of times, and the particular topic to be discussed here is one regarding which specialists have been at loggerheads for over thirty years. I include it because MiFRA is incomplete without it, but it may be skipped without loss of continuity. 143
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A Challenge from Textbook Bioenergetics and <strong>Free</strong> <strong>Radical</strong> Chemistry<br />
Fig. 11.2. Variation <strong>of</strong> pH near a phospholipid membrane.<br />
big (as are nearly all proteins) to fit through the pores in the outer membrane and the<br />
mitochondrial version is similarly trapped by the inner membrane. <strong>The</strong>refore, the H2O2<br />
formed in the intermembrane space is that formed by non-enzymatic dismutation, which<br />
(as noted above) slows down when respiration slows down. This has been further confirmed<br />
by the histochemical detection <strong>of</strong> singlet oxygen in the intermembrane space; 24c recall from<br />
Table 3.3 that singlet oxygen is generated by the non-enzymatic, but not the enzymatic,<br />
dismutation reaction. 24d Most analyzes have ignored non-enzymatic dismutation and<br />
therefore presumed that the H2O2 in the intermembrane space gets there by diffusion from<br />
either the cytosol or the matrix (where it was formed by superoxide dismutase); but if that<br />
is wrong, then the overall level <strong>of</strong> H2O2 may well fall when respiration slows, even if the level<br />
<strong>of</strong> superoxide rises. Yet another reason why this is so derives from the presence in the<br />
intermembrane space <strong>of</strong> cytochrome c, which (when in the ferric state) assiduously detoxifies<br />
superoxide back to oxygen by accepting its lonely electron, 25 but which also (when in<br />
the ferrous state) just as readily 26 donates an electron to HO2•, forming—yes—H2O2. This<br />
too would, therefore, happen at a slower rate if there were less HO2• present.<br />
Again it may seem as though I have comprehensively rebutted the challenge with which<br />
this chapter began. Again, the truth is very different.<br />
11.3. Mitchell’s Oversimplification<br />
I may as well warn the reader honestly, in advance, that this section is the most arcane<br />
in the book. Bioenergetics is a challenging discipline at the best <strong>of</strong> times, and the particular<br />
topic to be discussed here is one regarding which specialists have been at loggerheads for<br />
over thirty years. I include it because MiFRA is incomplete without it, but it may be skipped<br />
without loss <strong>of</strong> continuity.<br />
143