A Metabolic Enzyme That Rapidly Produces Superoxide, Fumarate Reductase of Escherichia coli

James A Imlay

doi:10.1016/s0021-9258(18)94548-5

Abstract

Aerobic organisms synthesize superoxide dismutases in order to escape injury from endogenous superoxide. An earlier study of Escherichia coli indicated that intracellular superoxide is formed primarily by autoxidation of components of the respiratory chain. In order to identify those components, inverted respiratory vesicles were incubated with five respiratory substrates. In most cases, essentially all of the superoxide was formed through autoxidation of fumarate reductase, despite the paucity of this anaerobic terminal oxidase in the aerobic cells from which the vesicles were prepared. In contrast, most dehydrogenases, the respiratory quinones, and the cytochrome oxidases did not produce any detectable superoxide. The propensity of fumarate reductase to generate superoxide could conceivably deluge cells with superoxide when anaerobic cells, which contain abundant fumarate reductase, enter an aerobic habitat. In fact, deletion or overexpression of the frd structural genes improved and retarded, respectively, the outgrowth of superoxide dismutase-attenuated cells when they were abruptly aerated, suggesting that fumarate reductase is a major source of superoxide in vivo. Steric inhibitors that bind adjacent to the flavin completely blocked superoxide production, indicating that the flavin, rather than an iron-sulfur cluster, is the direct electron donor to oxygen. Since the turnover numbers for superoxide formation by other flavoenzymes are orders of magnitude lower than that of fumarate reductase (1600 min-1), additional steric or electronic factors must accelerate its autoxidation.

Highlights

This enzyme maintains intracellular superoxide levels around 10- 10 M [1]
The number of molecules of superoxide produced per unit of electron flux through the respiratory chain depended upon the identity of the respiratory substrate
The small amount of 0; that was produced with saturating inhibitor either represents a slight degree of continued flavin exposure or some 0; production at a second site. Because these inhibitors bind to Fumarate reductase (Frd) and succinate dehydrogenase (Sdh) across the flavin, these results indicate that the flavin must be the site of electron leakage to oxygen

Summary

Introduction

This enzyme maintains intracellular superoxide levels around 10- 10 M [1]. Mutant strains of Escherichia coli, yeast, and Drosophila that lack SOD accumulate superoxide to much higher levels and exhibit severe growth deficits [2,3,4]. The instability of the NAD· species [12] prohibits NADH from spontaneously transferring electrons to oxygen On this basis, it is reasonable to consider flavin and quinone moieties as potential superoxide sources, since both form stable semiquinone species when univalently oxidized. Because respiration directs a large electron flux through a series of flavins, quinones, ironsulfur clusters, and heme groups, it is widely suspected that the respiratory chain may be the primary source of superoxide in aerobic organisms. This expectation was supported by an investigation of superoxide production by lysates of E. coli, which demonstrated that the particulate fraction generated more superoxide than did cytosolic enzymes [1]. The present work identifies the respiratory enzyme fumarate reductase of E. coli as a active source of superoxide

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