Oxygen: The dark side.

Irwin Fridovich Department of Biochemistry Duke University Medical Center Durham, NC 27710 Aerobes certainly gain advantages from 02; in genera- tion of useful energy, in intermediary metabolism, and in detoxication of hydrophobic xenobiotics. But, there is a down side. 02 is a paramagnetic gas, by virtue of containing two unpaired electrons, and this predisposes it to reduction in univalent steps. Intermediates are encountered on this univalent pathway; which are reac- tive enough to rend the delicate fabric of living sub- stance. These are the superoxide radical (02-), hydro- gen peroxide (H202) , and the hydroxyl radical (HO'). Furthermore, 02- can give rise to H202 by dismutation, either spontaneous or catalyzed; and 02- plus H202 can conspire to produce HO.. This production of HO~ occurs in vitrowhen 02- reduces Fe(lll) to Fe(ll); which in turn reduces H202 to OH- plus HOo. The in vivo process is quite different because 02 - then participates by oxidizing the [4Fe-4S] clusters of dehydratases, such as aconi- tase. The oxidized cluster is unstable and loses Fe(ll) which is then able to reduce H202 to OH- plus HO. (1,2). These reactive progenies of oxygen reduction are really produced under ordinary circumstances in aerobic cells, in amounts which would be intolerable were it not for multilayered defenses. These are superoxide dismutases, to convert 02 into H202 + 02, and catalases to convert H202 into 02 + H20. There are also peroxi- dases which use cellular reductants, such as G-SH and NADPH, to reduce H202 to water, and alkyl hydroperox- ides to corresponding alcohols. These defenses mark- edly diminish the damage which is done by reactive oxygen-derived species, but they do not entirely prevent such damage. This necessitates repair and resynthesis and there are enzymes dedicated to these tasks. Unreliable Measures of 02. Bis-acrydyllium salts (lucigenin) can be univalently reduced to a monocation radical, which adds to 02- to yield an unstable dioxetane, whose decomposition results in luminescence. Since this chemiluminescence depends upon 02 - it is inhibited by SOD and can be used to assay SOD activity, both in vitro (3) and within living cells (4). Lucigenin lumines- cence has also been used as a measure of 02-; but this is not advisable because the lucigenin monocation radi- cal can autoxidize and produce 02. Any system capable of the univalent reduction of lucigenin would thus appear to be producing O2-; even when it could not do so in the absence of lucigenin. Tetrazolium salts can similarly mediate 02- production and give a false indication of 02- production (5). Amino phthalhydrazide (luminol) lumi- nescence is also a misleading indicator of 02- produc- tion. In this case the luminol is oxidized univalently to a radical, which can reduce 02 to O2-; or which can add 02- to yield an unstable endoperoxide whose decomposition leads to luminescence (6). Bacterial Cu, ZnSODs - It has become apparent that gram negative bacteria commonly contain a periplasmic Cu,ZnSOD. In the case of E. colithis SOD appears late in the growth cycle (7) and is peculiar in being mono- meric; whereas most Cu,ZnSODs described to date have been homodimeric. The periplasmic localization suggests that this SOD may protect against exogenous O2. It certainly contributes to survival in stationary phase (8). 4.