Mechanism of superoxide and hydrogen peroxide formation by fumarate reductase, succinate dehydrogenase, and aspartate oxidase.

Oxidative stress is created in aerobic organisms when molecular oxygen chemically oxidizes redox enzymes, forming superoxide (O2*-) and hydrogen peroxide (H2O2). Prior work identified several flavoenzymes from Escherichia coli that tend to autoxidize. Of these, fumarate reductase (Frd) is notable both for its high turnover number and for its production of substantial O2*- in addition to H2O2. We have sought to identify characteristics of Frd that predispose it to this behavior. The ability of excess succinate to block autoxidation and the inhibitory effect of lowering the flavin potential indicate that all detectable autoxidation occurs from its FAD site, rather than from iron-sulfur clusters or bound quinones. The flavin adenine dinucleotide (FAD) moiety of Frd is unusually solvent-exposed, as evidenced by its ability to bind sulfite, and this may make it more likely to react adventitiously with O2*-. The autoxidizing species is apparently fully reduced flavin rather than flavosemiquinone, since treatments that more fully reduce the enzyme do not slow its turnover number. They do, however, switch the major product from O2*- to H2O2. A similar effect is achieved by lowering the potential of the proximal [2Fe-2S] cluster. These data suggest that Frd releases O2*- into bulk solution if this cluster is available to sequester the semiquinone electron; otherwise, that electron is rapidly transferred to the nascent superoxide, and H2O2 is the product that leaves the active site. This model is supported by the behavior of "aspartate oxidase" (aspartate:fumarate oxidoreductase), an Frd homologue that lacks Fe-S clusters. Its dihydroflavin also reacts avidly with oxygen, and H2O2 is the predominant product. In contrast, succinate dehydrogenase, with high potential clusters, generates O2*- exclusively. The identities of enzyme autoxidation products are significant because O2*- and H2O2 damage cells in different ways.

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