The identification of primary sites of superoxide and hydrogen peroxide formation in the aerobic respiratory chain and sulfite reductase complex of Escherichia coli

The fitness of organisms depends upon the rate at which they generate super oxide (O-2(radical anion)) and hydrogen peroxide (H2O2) as toxic by-product s of aerobic metabolism. In Escherichia coli these oxidants arise primarily from the autoxidation of components of its respiratory chain. Inverted ves icles that were incubated with NADH generated O-2(radical anion) and H2O2 a t accelerated rates either when treated with cyanide or when devoid of quin ones, implicating an NADH dehydrogenase as their source. Null mutations in the gene encoding NADH dehydrogenase II averted autoxidation of vesicles, a nd its overproduction accelerated it. Thus NADH dehydrogenase II but not NA DH dehydrogenase I, respiratory quinones, or cytochrome oxidases formed sub stantial O2(radical) (anion) and H2O2. NADH dehydrogenase II that was purif ied from both wild-type and quinone-deficient cells generated similar to 13 0 H2O2 and 15 O-2(radical anion) min(-1) by autoxidation of its reduced FAD cofactor, Sulfite reductase is a second autoxidizable electron transport c hain of E, coli, containing FAD, FMN, [4Fe-4S], and siroheme moieties, Puri fied flavoprotein that contained only the FAD and FMN cofactors had about t he same oxidation turnover number as did the holoenzyme, 7 min(-1) FAD(-1). Oxidase activity was largely lost upon FMN removal. Thus the autoxidation of sulfite reductase, like that of the respiratory chain, occurs primarily by autoxidation of an exposed flavin cofactor. Great variability in the oxi dation turnover numbers of these and other flavoproteins suggests that endo genous oxidants will be predominantly formed by only a few oxidizable enzym es. Thus the degree of oxidative stress in a cell may depend upon the titer of such enzymes and accordingly may vary with growth conditions and among different cell types. Furthermore, the chemical nature of these reactions w as manifested by their acceleration at high temperatures and oxygen concent rations. Thus these environmental parameters may also directly affect the O 2(radical anion) and H2O2 loads that organisms must bear.