Messner Kevin R, Imlay James A
Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 2002 Nov 8;277(45):42563-71. doi: 10.1074/jbc.M204958200. Epub 2002 Aug 27.
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.
当分子氧对氧化还原酶进行化学氧化时，需氧生物体内就会产生氧化应激，形成超氧化物（O2*-）和过氧化氢（H2O2）。先前的研究确定了大肠杆菌中几种易于自氧化的黄素酶。其中，延胡索酸还原酶（Frd）不仅因其高周转数引人注目，还因其除了产生H2O2外还会产生大量的O2*-。我们试图确定Frd导致这种行为的特征。过量琥珀酸能够阻止自氧化，以及降低黄素电位的抑制作用表明，所有可检测到的自氧化都发生在其FAD位点，而非来自铁硫簇或结合醌。Frd的黄素腺嘌呤二核苷酸（FAD）部分异常地暴露于溶剂中，这可通过其与亚硫酸盐结合的能力得到证明，这可能使其更易与O2*-发生偶然反应。自氧化物种显然是完全还原的黄素而非黄素半醌，因为能使酶更完全还原的处理并不会降低其周转数。然而，这些处理确实会将主要产物从O2*-转变为H2O2。降低近端[2Fe-2S]簇的电位也能产生类似效果。这些数据表明，如果该簇可用于隔离半醌电子，Frd就会将O2*-释放到大量溶液中；否则，该电子会迅速转移至新生的超氧化物，而H2O2则是离开活性位点的产物。“天冬氨酸氧化酶”（天冬氨酸：延胡索酸氧化还原酶）的行为支持了这一模型，它是一种缺乏铁硫簇的Frd同源物。其二氢黄素也能与氧气剧烈反应，且H2O2是主要产物。相比之下，具有高电位簇的琥珀酸脱氢酶则仅产生O2*-。酶自氧化产物的特性具有重要意义，因为O2*-和H2O2以不同方式损伤细胞。
