©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Metabolic Enzyme That Rapidly Produces Superoxide, Fumarate Reductase of Escherichia coli(*)

(Received for publication, April 6, 1995; and in revised form, June 8, 1995)

James A. Imlay (§)

From the Department of Microbiology, University of Illinois, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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), additional steric or electronic factors must accelerate its autoxidation.


INTRODUCTION

Synthesis of superoxide dismutase (SOD(^1); EC 1.15.1.1; ) is ubiquitous among aerobic and aerotolerant organisms.

This enzyme maintains intracellular superoxide levels around 10M(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) . In humans, dominant mutations in the structural gene encoding cytosolic SOD are associated with the development of familial amyotrophic lateral sclerosis(5) .

Recent studies in E. coli have identified some important physiological targets of superoxide. Among these are aconitase(6) , dihydroxyacid dehydratase(7) , fumarases A and B(8) , and 6-phosphogluconate dehydratase(9) , all of which belong to a class of dehydratases whose substrate-liganding iron-sulfur clusters disintegrate upon oxidation by superoxide. Second-order rate constants for these damaging reactions can exceed 10^6M s(10) . The inactivation of these enzymes is responsible for at least some of the phenotypic deficits of SOD-deficient mutants of E. coli.

Because superoxide cannot traverse membranes(11) , the superoxide that damages those enzymes in SOD-deficient mutants is evidently formed inside the boundary of the cytoplasmic membrane. The goal of this work is to identify the predominant mechanisms of its formation. Superoxide is produced when a single electron is transferred from a donor molecule to molecular oxygen. The E`(o) for univalent oxygen reduction is low (-0.16 V), requiring that a donor be a strong univalent reductant in order to push the reaction forward. Most biological electron donors do not meet this standard. For example, the instability of the NADbullet 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. Metal centers are similarly facile at single-electron redox reactions. Because respiration directs a large electron flux through a series of flavins, quinones, iron-sulfur 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 precise sites of electron leakage to oxygen were not identified in those experiments. Various studies with mitochondria have implicated autoxidation of ubiquinone and/or respiratory dehydrogenases(13) . The present work identifies the respiratory enzyme fumarate reductase of E. coli as a particularly active source of superoxide.


MATERIALS AND METHODS

Strains and Media

Strains and plasmids used in this study are described in Table 1. LB medium contained 10 g of bactotryptone, 5 g of yeast extract, 10 g of NaCl, and 2 g of glucose per liter. Defined media contained minimal A salts (15) plus carbon sources and thiamine. Casamino acids medium contained (per liter) 10 g of casamino acids; glucose/amino acids medium, 5 g of glucose, and 1.5 g of casamino acids; succinate medium, 25 mM succinate; and glycerol/fumarate medium, 0.5 g of casamino acids, 40 mM glycerol, and 40 mM fumarate. Plasmid pH3 was maintained with 60 µg/ml ampicillin; and plasmid pGS133 was maintained with 100 µg/ml kanamycin, since this concentration reportedly assists in overexpression of the enzyme(14) .



Uracil (0.2 mM) was supplemented to anaerobic cultures of quinone-deficient strains in order to by-pass the requirement for function of the respiration-linked dihydroorotate dehydrogenase. When vesicles were prepared from aerobic Ubi Men strains, pre-cultures were grown anaerobically (without 4-hydroxybenzoic acid) in order to avoid outgrowth of any Ubi revertants, and cultures were shifted to air-saturated medium two to three generations prior to harvesting. The frequency of reversion was checked by streaking the culture at the time of harvest onto LB medium; revertants, which form large colonies, always comprised <0.1% of the harvested bacteria.

Genetic Techniques

P1-mediated transduction and plasmid transformation were conducted according to standard methods(15) . To prevent reversion of the ubiA420 allele during strain constructions, 1 mM 4-hydroxybenzoic acid was supplemented to the growth medium in order to allow some ubiquinone biosynthesis(16) . The presence of the sdhC4 and Delta(frdABCD)18 null alleles, respectively, were confirmed by the inability of such mutants to grow in aerobic succinate medium or anaerobic glycerol/fumarate medium, respectively. The sdhC4 null allele in quinone-deficient strains was verified by enzymatic assay of succinate:plumbagin oxidoreductase activity (see below), and the presence of the Delta(frdABCD)18 mutation was demonstrated in quinoneless backgrounds by retransducing the linked Tet^r marker from the putative mutant into AB1157 and confirming that these secondary transductants had lost the ability to grow in anaerobic glycerol/fumarate medium.

Preparation of Inverted Vesicles

Aerobic cultures were grown with vigorous shaking for at least five generations before being harvested at approximately 1 10^8 cells/ml (A = 0.3). Measurements made with a Clarke electrode verified that the cultures were fully air-saturated at this density. Anaerobic cultures were grown in an anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI) under 85% N(2), 5% CO(2), 10% H(2) and monitored spectrophotometrically throughout the growth period to ensure that cultures were growing exponentially up to the point of harvesting. To avoid induction of enzymes during the harvesting process, 150 µg/ml chloramphenicol was added 10 min before cultures were removed from the shaker or anaerobic chamber. Cultures were then chilled in ice water, centrifuged, washed with ice-cold 50 mM potassium P(i) (pH 7.8), resuspended the same buffer at about 0.5% the initial cell density, and lysed by passage through a French pressure cell. The lysate was clarified by centrifugation at 20,000 g for 20 min. The inverted membrane vesicles were then pelleted by centrifugation at 100,000 g for 2 h, resuspended in the same buffer, centrifuged again for 2 h, and finally suspended into about 0.2% the original culture volume. This procedure eliminated all detectable superoxide dismutase activity. Vesicles were stored on ice. With the exception of NADH dehydrogenase I, the dehydrogenase and superoxide-production activities declined by <10% over a week. NADH dehydrogenase I is very unstable during storage on ice (17) and was virtually inactive in the experiments reported here.

Assays for Superoxide Production

Cytochrome c Reduction

Superoxide formation was quantitated as SOD-sensitive cytochrome c reduction(1) . Inverted vesicles were incubated in 3 ml of air-saturated 50 mM potassium P(i) (pH 7.8) at the indicated temperatures in the presence of 10 µM ferricytochrome c. Respiratory substrates, also at pH 7.8, were added; if present, KCN was 3.3 mM. Consequent cytochrome c reduction was quantitated by absorbance measurements at 550 nm with a Beckman DU600 UV-visible spectrophotometer using the relation cyc - cyc = 21.0. In a parallel reaction 20 units of manganese-containing superoxide dismutase (which is not inhibited by cyanide) was included. The superoxide yield was then calculated by subtraction of the superoxide dismutase-resistant absorbance change from the total absorbance change.

The cytochrome c assay was used to measure superoxide production by xanthine oxidase both in the presence and absence of vesicles. There was no difference, even at very high vesicle concentrations (data not shown). This was also true when respiratory substrates were included to stimulate electron flow through the respiratory chain. Thus no evidence was observed to support the possibility (18) that either reduced or oxidized respiratory chain components are effective scavengers of superoxide.

Epinephrine Oxidation

The oxidation of epinephrine by superoxide (19) was measured in 3-ml reactions containing vesicles, respiratory substrate, and 0.5 mM epinephrine. Reactions were buffered with 50 mM potassium P(i) (pH 7.8), as used in the cytochrome c system above. At this pH the oxidation of epinephrine is strictly dependent upon an enzymatic superoxide source. Epinephrine oxidation was monitored by the absorbance at 480 nm of its product, adrenochrome, and was almost fully inhibitable by 400 units of SOD. The superoxide yield from respiratory enzymes was quantitated ( = 4.0) after calibration of the detection system using xanthine oxidase, whose superoxide production activity had previously been determined by the cytochrome c method. The four-electron oxidation of epinephrine by O occurs by a chain reaction, the length of which depends upon epinephrine concentration and buffer composition. With both xanthine oxidase and respiratory enzymes the ratio of superoxide-dependent cytochrome c reduction to adrenochrome formation was 1.58 ± 0.04, indicating that the chains are of identical length and justifying the use of this technique as a quantitative assay of O.

Lucigenin Luminescence

Luminometry was performed in a Turner Model 20e luminometer (Turner Designs, Mountain View, CA). One-ml reactions contained 100 µM lucigenin, vesicles, and substrate in 50 mM glycine buffer (pH 9.0). Reactions were initiated by the addition of substrate, and data were collected and summed over the subsequent 10 s. Light emission with any superoxide source was completely quenched at pH 7.8, requiring that reactions be run at the higher, nonphysiological pH.

Assays of Respiratory Oxidase and Dehydrogenase Activities

Oxidase activities were measured in potassium P(i) buffer with a Clarke electrode. Dehydrogenase activities were assayed using 0.4 mM of the naphthoquinone plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) to mediate electron transfer from the dehydrogenase to cytochrome c, in the presence of 3.3 mM KCN(20) . Ferricyanide reductase measurements were made with 1 mM ferricyanide added to vesicles in the presence of 3.3 mM KCN and saturating reductive substrate (21) using = 1.0 at 420 nm for ferricyanide. In order to calculate turnover numbers, succinate dehydrogenase was quantitated by absorbance change of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide in the presence of phenazine methosulfate exactly as described(14) . Fumarate reductase was quantitated for the same purpose using the phenazine methosulfate-mediated reduction of dichlorophenolindophenol(22) , with extrapolation to determine the rate at infinite concentration of the mediator, as described(21) .

To verify the absence of quinones from vesicles prepared from Ubi Men strains, NADH oxidase activity was measured and compared to NADH dehydrogenase activity. The ratio of NADH oxidase activity per NADH dehydrogenase capacity in quinoneless vesicles was approximately 1% that of wild-type vesicles, confirming the virtual or complete absence of endogenous ubiquinone and menaquinone.

Determination of Kof Fumarate Reductase for Malonate and Malate

Inverted vesicles were prepared from AB1157 after anaerobic growth in glycerol/fumarate medium. The succinate:plumbagin oxidoreductase activity of fumarate reductase was determined at 12 concentrations between 20 µM and 40 mM succinate as described above, using 0.4 mM plumbagin, which is virtually saturating even at 80 mM succinate. The effects of competitive inhibitors were determined at four concentrations of inhibitor, and K values were calculated by Lineweaver-Burk analysis. To determine apparent K values for blockage of O formation, vesicles were incubated with 20 mM alpha-glycerolphosphate. In this circumstance electrons are transferred from the alpha-glycerolphosphate dehydrogenase through the quinone pool to Frd, which autoxidizes at a high rate. Rates of O production at the fumarate reductase site were measured in the presence of inhibitors. Inhibitors did not lessen alpha-glycerolphosphate:plumbagin oxidoreductase activity, indicating that they did not inhibit the alpha-glycerolphosphate dehydrogenase. Frd did not exhibit any malonate:plumbagin or malate:plumbagin oxidoreductase activity, demonstrating that neither inhibitor was redox-active with the enzyme.

Miscellaneous

Measurements of succinate dehydrogenase behavior were conducted after a 10 min preincubation with succinate at 37 °C to displace inhibitory oxaloacetate. Xanthine oxidase was used to generate superoxide with 50 µM xanthine in the standard potassium P(i) medium. Xanthine oxidase activity was independently assayed by monitoring urate production at 295 nm [Delta(xanthine to urate) = 11.0]. Superoxide dismutase was assayed using the xanthine oxidase/cytochrome c method(23) . The production of H(2)O(2) was quantitated using the horseradish peroxidase-coupled oxidation of o-dianisidine(24) . Protein was assayed with the Coomassie Dye reagent (Pierce).

Chemicals

B-NADH, succinic acid, DL-alpha-glycerolphosphatebullet2Na, D-lactic acid, E. coli manganese-containing superoxide dismutase, xanthine oxidase, xanthine, horse heart ferricytochrome c (type III), horseradish peroxidase (type II), plumbagin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, diethylenetriamine pentaacetic acid, phenazine methosulfate, 2,6-dichlorophenolindophenol, o-dianisidine, fumaric acid, malonic acid, racemic malic acid, oxaloacetic acid, potassium ferricyanide, lucigenin (bis-N-methylacribinium nitrate), Tris, MOPS, chloramphenicol, ampicillin, kanamycin sulfate, tetracycline hydrochloride, acid-hydrolyzed casamino acids, vitamins, and uracil were purchased from Sigma. Potassium cyanide, 4-hydroxybenzoic acid, L-lactic acid, and (R)-(-)-epinephrine were from Aldrich; monobasic and dibasic potassium phosphate salts, from EM Science. Water used for all in vitro experiments was house-deionized water further purified by passage through a Labconco Water Pro PS system.


RESULTS

Superoxide Is Produced during Respiration in Vitro

Inverted respiratory vesicles were prepared from wild-type E. coli after aerobic growth in casamino acids medium. This medium was chosen because it promotes synthesis of a wide variety of respiratory dehydrogenases(25) . Accordingly, when incubated in air-saturated buffer, the isolated membranes readily oxidized each of the respiratory substrates NADH, succinate, D-lactate, L-lactate, and alpha-glycerolphosphate. The concomitant production of superoxide was assayed by its ability to reduce cytochrome c, as described under ``Materials and Methods,'' and the calculated superoxide yields are presented as Table 2.



The number of molecules of superoxide produced per unit of electron flux through the respiratory chain depended upon the identity of the respiratory substrate. In general, approximately 0.5% of the respiratory oxygen consumption was due to superoxide formation.

With all substrates the rate of superoxide production in vitro was linear with time (Fig. 1A) and increased in proportion to vesicle concentration (not shown). However, high substrate concentrations actually inhibited superoxide production when respiration was driven by succinate (Fig. 1B). This effect is not due to interference with the superoxide detection system, since succinate did not affect the ability of cytochrome c to be reduced by superoxide that was generated by xanthine oxidase. This phenomenon is explored in further detail below.


Figure 1: A, time course of superoxide production by inverted vesicles. Vesicles were prepared from an aerobic culture of AB1157 and incubated with 0.3 mM succinate. B-D, superoxide production versus succinate concentration. Vesicles prepared from cultures grown in aerobic casamino acids medium were incubated at 23 °C for 7.5 min with the indicated concentrations of succinate. The superoxide yield is presented per vesicles from 10 bacteria in order to enable comparisons among the three strains. Note that the abscissa is drawn on an exponential scale. Circles, no additions. Squares, 3.3 mM cyanide present. B, superoxide from vesicles of AB1157 (Sdh Frd). Dashed curve, no additions, with signal expanded 7.5-fold so that the profile can be compared to that obtained in the presence of cyanide. Triangles, rate of superoxide production by xanthine oxidase, determined by the cytochrome c method. C, superoxide from vesicles of JI241 (Sdh Frd). D, superoxide from vesicles of JI222 (Sdh Frd).



Superoxide Does Not Escape from the Terminal Oxidases during the Reduction of Oxygen to Water

The purpose of this study was to identify sites on the respiratory chain that rapidly generate superoxide. A modular diagram of the aerobic chain is shown in Fig. 2A. Since all of the respiratory substrates stimulated the production of superoxide, the most economical hypothesis was that the superoxide was evolved by either the quinones or the cytochrome oxidases, since electrons pass through these carriers no matter what the substrate. In particular, one might imagine that superoxide occasionally escapes from the cytochrome oxidases as a partially reduced intermediate in the four-electron reduction of molecular oxygen to water. To test this possibility, cyanide, which binds to the cytochrome oxidases and prevents their association with molecular oxygen, was added to the in vitro assays of superoxide production. The effect of the cyanide was actually to increase the rate of superoxide production, with each respiratory substrate (Table 2). These data indicate that superoxide was formed when electrons were leaked to molecular oxygen by a component upstream of the cytochrome oxidases; the acceleration by cyanide was presumably due to the greater electron occupancy on that component, since outflow was blocked. A similar enhancement of superoxide production occurs when downstream inhibitors are added to respiring submitochondrial particles (13) .


Figure 2: Modular diagram of the respiratory chain. Vertical axis denotes relative free energies of electron carriers; exact relationships depend upon the relative abundance and redox status of the carriers. For simplicity a single cytochrome oxidase and a single alpha-glycerolphosphate dehydrogenase are depicted, and anaerobic reductases induced by alternative electron acceptors are omitted. For a complete treatment see (43) and (44) . A, aerobic chain. B, anaerobic chain. Fumarate is acquired either by import from the growth medium (45) or by reversal of the terminal reactions of the TCA cycle.



Superoxide Is Formed by Autoxidation of Succinate- and NADH-reducible Dehydrogenases

Soluble ubiquinone analogues autoxidize in vitro and in vivo, and some studies of mammalian mitochrondria have suggested that the spontaneous autoxidation of reduced ubiquinone might comprise an important source of superoxide(18) . Membranes were prepared from strains that lacked quinones due to mutations in both the ubiquinone and menaquinone biosynthetic pathways. The poor-growth phenotype affected the relative abundance of some dehydrogenases, reducing the alpha-glycerolphosphate dehydrogenase and the lactate dehydrogenase activities to virtually undetectable levels and obviating measurements of superoxide formation during incubation with these substrates. However, the succinate and NADH dehydrogenases were present, and superoxide was produced at undiminished rates when the quinoneless vesicles were incubated with either of these substrates (data not shown). Since the electrons could not move beyond the enzymes which succinate and NADH directly reduced, the superoxide must have been generated by autoxidation of those enzymes themselves. E. coli expresses two respiratory NADH dehydrogenases; preliminary data obtained in this laboratory suggests that NADH dehydrogenase II was responsible for the superoxide detected in these experiments and leaves open the question of whether NADH dehydrogenase I produces superoxide. The remainder of this report focuses upon the site of superoxide production during respiration of succinate.

Fumarate Reductase Is a Major Source of Superoxide

The experimental results discussed above suggested that succinate dehydrogenase (Sdh) was the probable source of superoxide during succinate-driven respiration. To test this prediction, vesicles were prepared from a mutant strain devoid of Sdh activity. Surprisingly, these vesicles evolved just as much superoxide as did those from its Sdh-proficient parent strain (Fig. 1C).

Fumarate reductase (Frd) is a second succinate-reducible enzyme associated with the respiratory chain. Frd acts as a terminal oxidase during anaerobic respiration (Fig. 2B), transferring electrons from the low-potential carrier menaquinone to fumarate, generating succinate as a final product. However, in vitro Frd can be directly reduced by succinate in a reversal of its physiological reaction. In fact, although in their physiological roles Frd and Sdh catalyze the interconversion of succinate and fumarate in opposite directions, they are structurally similar isozymes, and the plasmid-driven aerobic synthesis of fumarate reductase can restore the ability to oxidize succinate in vivo to mutants that lack succinate dehydrogenase(26) . Because fumarate-directed respiration is energetically less profitable than oxygen-directed respiration, fumarate reductase is generally repressed during aerobiosis(27) . Thus it was unexpected that Frd might be present and responsible for superoxide production in these vesicles, which were prepared from cells that had been grown with vigorous aeration.

Yet vesicles prepared from frd deletion mutants failed to evolve superoxide when incubated with succinate (Fig. 1D), demonstrating that Frd was indeed the site of succinate-dependent superoxide production. The addition of cyanide to the Frd-deficient membranes restored some superoxide formation, and even this amount was absent from vesicles lacking both Frd and Sdh (not shown). Thus Sdh transfers electrons to molecular oxygen, producing superoxide, only when the usual electron flow down the respiratory chain is blocked; in unblocked vesicles oxidized ubiquinone outcompetes oxygen for electrons on the reduced enzyme. It therefore seems that Frd, but not Sdh, may be an important source of superoxide in vivo.

The apparent presence of Frd in membranes prepared from aerobic cultures appeared to contradict reports that its synthesis is almost fully repressed during aerobiosis(27) . Measures had been taken to ensure that the exponential cultures remained air-saturated until they were harvested and that protein synthesis did not occur under the microaerobic conditions that occur during subsequent centrifugation (``Materials and Methods''). Frd content and superoxide formation were therefore quantitated in membranes derived from cells grown both in casamino acids medium and in the glucose media used by other workers (Table 3). It is apparent that casamino acids promote the aerobic synthesis of Frd, although the basis of this induction is unknown. These levels were still far below those which were achieved in anaerobic media, when Frd can fulfill its physiological role of serving as an alternate terminal oxidase. In all preparations the rate of succinate-dependent superoxide production paralleled the Frd content of the membranes (Table 3). Furthermore, 7-fold overexpression of Frd from plasmid pH3 caused an 8-fold increase in superoxide production by vesicles prepared from JI241.



The epinephrine-oxidation and lucigenin assays of O production quantitatively confirmed the cytochrome c data (Table 4). These results verify that Frd is a highly active source of superoxide.



Frd Is Responsible for O Production during Respiration of Varied Substrates

Because Frd is situated in the respiratory chain as a terminal oxidase, it receives from the quinone pool electrons that originated from a variety of respiratory substrates. Thus, because of its predisposition to transfer electrons to oxygen, Frd could conceivably have been the source of the superoxide that was detected when vesicles were incubated with respiratory substrates other than succinate.

In accord with this idea, vesicles that were prepared from anaerobic cells, which have abundant Frd, generated far more respiratory O than did vesicles from aerobic cells, no matter what the respiratory substrate (Table 5). In contrast, vesicles prepared from Frd-deficient mutants did not generate detectable amounts of O with alpha-glycerolphosphate, D-lactate, or L-lactate. The essential role of Frd in generating O with these substrates was also confirmed by the fact that the inclusion in the assay of fumarate, which re-oxidizes Frd and thereby prevents electron transfer to oxygen, prevented O formation with these substrates. Oxygen-directed respiration continued in the presence of fumarate, so the absence of O production indicates that these dehydrogenases, the quinones, and the terminal oxidases are not significant sources of O. The lone exception was NADH, which generated some O through autoxidation of the NADH dehydrogenase. In anaerobically derived vesicles, however, even the contribution of NADH dehydrogenase to superoxide formation was overshadowed by that of Frd.



Electrons are delivered to Frd by the low-potential carrier menaquinone, and vesicles derived from mutants lacking menaquinone did not generate any O at Frd when incubated with alpha-glycerolphosphate, L-lactate, or NADH (not shown). Table 5shows that those substrates whose dehydrogenases are most kinetically competent at menaquinone reduction, alpha-glycerolphosphate, D-lactate, and, to a lesser degree, NADH(28) , were those which stimulated the most O production by Frd.

Thus, when present, Frd was the source of virtually all of the respiratory O. The tendency of this enzyme to produce O is evidently not shared by the other respiratory dehydrogenases or oxidases.

Frd Generates O in Vivo

The abundance of Frd in anaerobic cells led to the prediction that Frd would produce much intracellular O when such cells were abruptly shifted into aerobic medium. Although no physical technique is currently available that can directly measure intracellular concentrations of O, the presence of abundant O in E. coli can be inferred from its inhibitory effects upon growth. In particular, excess O inactivates dihydroxyacid dehydratase (7) and thereby prevents growth in the absence of branched-chain amino acid supplements (2) .

When wild-type cells that had been cultured in anaerobic glucose minimal medium were aerated, they continued to grow without significant delay (Fig. 3A). Evidently the concentration of intracellular O was insufficient to block amino acid biosynthesis. In this circumstance the O concentration is suppressed primarily by action of the iron-containing superoxide dismutase, which is synthesized pre-emptively during anaerobiosis in preparation for re-exposure to oxygen. In contrast, the growth of sodB mutants, which lack that enzyme, lags approximately 70 min after re-aeration, finally resuming when the oxygen-inducible manganese-containing superoxide dismutase accumulates in sufficient quantity to reduce endogenous O to subtoxic levels ( (29) and Fig. 3A). To determine whether Frd was the primary source of the toxic O, strains lacking both Fe-SOD and Frd were constructed. Upon aeration the sodB frd mutants lagged more briefly than did those containing Frd. Conversely, strains carrying a multicopy plasmid encoding Frd exhibited an increase in growth lag (Fig. 3B).


Figure 3: Correlation between Frd content and oxygen toxicity upon reaeration of SOD-deficient strains. A, strains AB1157 (SOD Frd), JI131 (sodB Frd), JAC4 (sodB frdA26), and YK100 (sodB DeltafrdABCD) were cultured into log-phase in anaerobic minimal glucose medium. At time 0, the cultures were diluted into aerobic medium of the same composition, and growth was monitored by absorbance at 600 nm. The outgrowth of all strains proceeded without a lag when branched-chain amino acids were provided in the culture medium (not shown). B, strains AB1157 (SOD Frd), JI131 (sodB Frd), JI314 (SOD with a plasmid overproducing Frd), and JI316 (sodB with a plasmid overproducing Frd) were cultured in anaerobic minimal medium and diluted at time 0 into aerobic medium of the same composition.



These data indicate that Frd may be a significant source of O in vivo. However, the residual lag of the frd sodB mutant indicates that sources of O other than Frd may be significant. Furthermore, the flux of O from Frd is not sufficient to debilitate an SOD-proficient cell. Possible reasons for this are explored under ``Discussion.''

Electron Transfer to Oxygen Occurs at the Flavin Sites of Both Frd and Sdh

Fumarate reductase and succinate dehydrogenase are both well characterized flavoenzymes. They each contain [3Fe-4S], [4Fe-4S], and [2Fe-2S] clusters. The flavin interacts directly with the succinate-fumarate couple, the [3Fe-4S] cluster exchanges electrons with the respiratory quinones, and the [2Fe-2S] cluster apparently bridges electron flow between them. The catalytic role, if any, of the very low-potential [4Fe-4S] cluster is not understood.

The kinetics of O production by Frd and Sdh were investigated in an effort to identify which of these redox moieties leaks electrons to oxygen. Excess succinate inhibited superoxide production in vitro by both Frd and Sdh, although the two enzymes exhibited somewhat different profiles (Fig. 1). This effect occurred both in respiring vesicles and when respiration was blocked by cyanide. To verify that this activity was a behavior of the isolated enzymes, Frd and Sdh were independently expressed in mutants devoid of quinones, preventing interaction between the enzymes and other components of the respiratory pathway. The enzymes recovered in vesicles from such cells continued to generate O when incubated at moderate, but not high, concentrations of succinate (Fig. 4). A similar profile was obtained when superoxide production was quantitated using an epinephrine-oxidation assay instead of cytochrome c reduction (Fig. 5). Yet excess succinate did not interfere with superoxide production or detection when enzymatic sources other than Sdh or Frd were used, including xanthine oxidase.


Figure 4: The succinate dependence of superoxide production by Frd and Sdh in quinone-deficient vesicles. A, succinate-dependent superoxide production by fumarate reductase. Vesicles were prepared from JI243 (Ubi Men Sdh Frd) after anaerobic growth on minimal glucose + casamino acids medium. Rates are each normalized to their maxima. Circles, superoxide production. Squares, reduction of 0.4 mM plumbagin. Diamonds, reduction of 1.0 mM ferricyanide. Maximum rates: 96 nmol/min superoxide production, 1.14 µmol/min plumbagin reduction, and 1.45 µmol/min ferricyanide reduction. B, succinate-dependent superoxide production by succinate dehydrogenase in quinone-deficient vesicles. Vesicles were prepared from JI332 (Ubi Men Frd with a plasmid overproducing Sdh) for assay of superoxide production or plumbagin reduction, and from AB1157 (Ubi Men Sdh) for assay of respiration. Circles, superoxide production. Maximum was 6.1 nmol/min/ml of vesicles. Rates below 1 µM succinate were unreliable due to succinate depletion during the period of measurement. Squares, reduction of 0.4 mM plumbagin. Maximum was 1.10 µmol/min/ml of vesicles. Triangles, respiration. In AB1157 electron transfer to plumbagin occurred at about 95% the maximal rate of respiratory oxygen consumption.




Figure 5: Kinetics of epinephrine oxidation by superoxide produced by fumarate reductase. Vesicles were prepared from strain JI243 (Ubi Men Sdh Frd). A, time course of adrenochrome accumulation. Reactions contained 10 µl of JI243 vesicles and 0.6 mM succinate. B, response to increasing amount of vesicles. Vesicles were incubated with 0.6 mM succinate for 15 min. C, circles, superoxide production by Frd as a function of succinate concentration. Each 7.5-min reaction contained 10 µl of JI243 vesicles; the maximum rate of adrenochrome formation was 58 nmol/min/ml of vesicles. This rate of superoxide production, calculated after xanthine oxidase was used to standardize the assay, was within 1% of that determined with the cytochrome c assay. Triangles, succinate does not prevent the detection of superoxide generated by xanthine oxidase. The inhibition evident when xanthine oxidase was incubated in >40 mM succinate was due to inhibition of the enzyme, as evidenced by a parallel decline in urate production.



The inhibition by concentrated succinate was immediately reversed when Frd-containing vesicles were diluted into lower concentrations of succinate, confirming that Frd was not irreversibly affected (data not shown). Interestingly, the high succinate concentrations did not suppress the rate of electron transfer by either enzyme to the artificial quinone plumbagin, and nor did they inhibit electron transfer from Sdh to endogenous ubiquinone (as measured by respiration rate) (Fig. 4). In fact, the superoxide-production optima were approximately coincident with the apparent K values for electron transfer to quinones. In summary, superoxide was maximally generated when the enzymes were half-saturated with substrate, and its formation was fully inhibited when they were saturated.

Two hypotheses might explain this effect. First, because both Frd and Sdh can accommodate a total of four electrons on their flavin and iron-sulfur clusters, it seemed possible that the half-reduced form of the enzymes might generate O, whereas the fully reduced forms could not. (The (4Fe-4S) cluster is not considered, since its low potential is not within the reach of the succinate-fumarate couple.) Such behavior is characteristic of xanthine oxidase, which exclusively forms H(2)O(2) instead of O when the fully reduced enzyme reacts with oxygen. Such a model would predict that the concentration of succinate optimal for O production would be lessened when respiratory blocks impeded electron outflow from Sdh. However, although the magnitude of O formation was affected, the optimal concentration of succinate was the same in both respiring and quinone-deficient membranes (Fig. 4). Furthermore, high concentrations of succinate did not accelerate H(2)O(2) production by these enzymes (data not shown).

The second possibility was that the binding of excess succinate to the face of the reduced enzyme might sterically obstruct interaction between oxygen and the autoxidizable redox center on the enzyme. If so, then the succinate analogues malonate and malate might also be expected to inhibit electron transfer from reduced Frd to oxygen. The avidity with which these analogues bind to the active site of Frd was demonstrated by their competitive inhibition of its succinate:plumbagin oxidoreductase activity, with apparent K values of 25 µM and 1.2 mM, respectively. Neither substrate was redox-active with Frd (``Materials and Methods'').

In the experiment presented in Fig. 6, Frd was reduced by the delivery of electrons from alpha-glycerolphosphate dehydrogenase via menaquinone. This route is responsible for the vast majority of O production during the respiration of alpha-glycerolphosphate (Table 4). The addition of either malonate or malate prevented O formation (Fig. 6). The kinetics of inhibition suggest K values of 110 µM for malonate and 2.5 mM for malate. These are somewhat higher than for the succinate:plumbagin oxidoreductase activity, presumably because of the greater affinity of these succinate analogues for the oxidized rather than the reduced forms of the enzyme. The small amount of O that was produced with saturating inhibitor either represents a slight degree of continued flavin exposure or some O production at a second site.


Figure 6: Structural analogues of succinate inhibit O production by fumarate reductase. A, experimental design. Menaquinone delivers electrons from alpha-glycerolphosphate dehydrogenase to the [3Fe-4S] cluster of Frd. Excess succinate [OOC-CH(2)-CH(2)-COO] or its analogues, malonate [OOC-CH(2)-COO] and malate [OOC-CH(2)-CH(OH)-COO], bind opposite the flavin of the enzyme. If excess succinate suppresses O production by shielding the flavin from oxygen, its competitive inhibitors may do so as well. Abbreviations: alpha-glyc-P, alpha-glycerolphosphate; DHAP, dihydroxyacetone phosphate; Glp, alpha-glycerolphosphate dehydrogenase; MQ, menaquinone. The respiratory enzymes are shown protruding from the inner face of the cytoplasmic membrane. B, inhibition of O production by malonate. C, inhibition of O production by a racemic mixture of DL-malate.



Because these inhibitors (and succinate) bind to Frd and Sdh across the flavin, these results indicate that the flavin must be the site of electron leakage to oxygen. Excess succinate does not block quinone reduction by either enzyme, because quinones dock at the distant [3Fe-4S] cluster.

Superoxide Turnover Numbers for Frd and Sdh

Tentative approximations of the superoxide turnover numbers for Frd and Sdh at 37 °C have been made using dye turnover numbers reported by other workers. The rates did not significantly differ when they were measured using MOPS or Tris as buffers in place of potassium P(i). Kita et al. (14) reported that Sdh reduces phenazine methosulfate with a turnover number of 1860 min. This assay was used to quantitate Sdh in the quinoneless vesicle preparation, and the turnover number for superoxide by the Sdh was then calculated to be approximately 13 min. Phenazine methosulfate reductase activity was measured in parallel with superoxide production for Frd, and a similar calculation was made using a turnover number of 15,850 min for electron transfer from Frd to phenazine methosulfate(22) . The turnover number of Frd for superoxide production was thereby calculated to be 1600 min. This substantially exceeds even that of xanthine oxidase (290 min(30) ); only the NADPH oxidase of neutrophils is known to generate superoxide so rapidly (Table 4). It is clear why all of the succinate-dependent superoxide production in aerobic membranes was due to Frd (Fig. 1), despite the far greater abundance of Sdh.

Superoxide Is Produced by the Native Forms of Frd and Sdh

Efforts were taken to establish that the superoxide is generated by native, undamaged forms of both Sdh and Frd. This consideration motivated the decision to study the enzymes in quinoneless membranes rather than in purified form, since inadvertent damage during purification or the exposure of Fe-S clusters normally buried in the lipid bilayer might cause the enzymes to acquire spurious autoxidation tendencies.

The following data indicated that the superoxide was generated by the native forms of both enzymes. 1) The superoxide-generating activity for both enzymes exhibited <10% loss during week-long storage on ice and was constant for at least 2 h during in vitro measurements. This stability argues against the possibility that the superoxide was produced by damaged forms of the enzymes that accumulate after cell lysis. 2) Superoxide production by both enzymes was completely inhibited by oxaloacetate (not shown), demonstrating that the superoxide is not generated by the artifactually oxaloacetate-bound form of the enzyme that can be recovered during vesicle preparation. 3) Superoxide production by both enzymes was prevented when fumarate was available to reoxidize the enzymes, indicating that the enzymes that generate the superoxide retain their fumarate reductase capacity. 4) The availability of ubiquinone also inhibited superoxide production by either enzyme, presumably by reoxidizing the enzyme before it could directly transfer electrons to oxygen. Thus the superoxide-producing enzymes retain their physiological succinate:ubiquinone oxidoreductase capacity. 5) Other workers have noted that flavin autoxidation can be catalyzed by loosely bound iron that contaminates biological buffers. That appears not to be the case for Frd and Sdh. The turnover numbers for superoxide production by Frd were essentially unchanged whether measured in Tris, MOPS, or phosphate buffers. Furthermore, the addition of up to 1 mM of the metal chelators EDTA and diethylenetriamine pentaacetic acid did not inhibit superoxide formation (data not shown). Collectively, these results argue strongly that the native enzymes are responsible for the generation of superoxide.


DISCUSSION

Sites of Superoxide Production

Most theories of oxygen toxicity assert that cell damage is caused by partially reduced oxygen species, either by superoxide directly or else by the H(2)O(2) and hydroxyl radicals that derive from it. Although superoxide has been presumed to arise during aerobic metabolism by autoxidation of reduced enzymes or electron carriers, it has been difficult to identify sources that are capable of generating superoxide at a substantial rate. Earlier studies indicated that the major source of endogenous superoxide in E. coli was the respiratory chain(1) . The results reported here indicate that superoxide evolves from the chain primarily by autoxidation of respiratory dehydrogenases, rather than as a by-product of terminal oxygen reduction or by quinone autoxidation. The quinone pool, in fact, has a antioxidant effect due to its ability to quickly remove electrons from the dehydrogenases before they are leaked to oxygen. The involvement of quinones in generating superoxide in mitochondria has been controversial(18) , in part due to the harshness of the treatments necessary to extract quinones from submitochondrial particles. However, most data indicate that at least some of the mitochondrial superoxide arises from the respiratory dehydrogenases(31) .

The rates at which the different respiratory enzymes produce superoxide vary widely. This work has unambiguously identified only Frd and NADH dehydrogenase II as significant sources of superoxide in respiring vesicles. L-Lactate dehydrogenase, D-lactate dehydrogenase, and alpha-glycerolphosphate dehydrogenase generated little or no superoxide directly. Interestingly, despite its similarities in structure and function to Frd, Sdh produced substantial superoxide only when respiration was artificially blocked, and even then only at a comparatively low rate. Thus the propensity of Frd to generate superoxide contrasts markedly to the behavior of most redox enzymes.

It is premature to suggest that the autoxidizability of the E. coli enzyme extends to fumarate reductases from other organisms. However, Turrens (32) reported that O was generated by the NADH-fumarate respiration of Trypanosoma brucei.

The Physiological Impact of Superoxide Production by Fumarate Reductase

When E. coli is grown in most aerobic media, very little Frd is made, and so it cannot comprise a significant source of superoxide. Instead, one might anticipate that superoxide production by Frd would acquire significance in the particular circumstance when anaerobic cells, which are laden with Frd, enter an air-saturated environment. This situation is an integral part of the lifestyle of facultative bacteria such as E. coli, occurring when the bacteria are excreted from the anaerobic colon into oxygenated surface waters. The abrupt exposure to oxygen of pre-formed Frd would be expected to cause a large flux of superoxide into the cytosol. Because electrons from any respiratory substrate can be directed from the quinone pool to Frd, this scenario would not be constrained to cells oxidizing succinate. Toxicity would arise because E. coli contains several critical enzymes that are rapidly inactivated by superoxide. Furthermore, in the presence of hydrogen peroxide, its dismutation product, superoxide accelerates the production of DNA damage. Thus the consequence of the autoxidizability of Frd might be that an oxidative crisis is forced upon the cell whenever it transits from an anaerobic to an aerobic habitat.

In fact, experiments with an SOD-deficient mutant supported the idea that Frd generates superoxide when anaerobic cells are re-aerated. However, it was clear that the bolus of superoxide was not sufficient to incapacitate SOD-proficient cells. This result was expected, since this facultative organism could not tolerate oxidative poisoning during every transit from an anaerobic to an aerobic environment. Survival upon aeration requires the preemptive synthesis of Fe-SOD when E. coli is anaerobic. Several additional factors may help minimize the superoxide flux from Frd. First, the resumption of ubiquinone synthesis upon aeration enables respiratory electrons to by-pass Frd as flow to cytochrome oxidase is restored. Furthermore, any Frd that manages nonetheless to receive electrons can be directly re-oxidized by ubiquinone before the electrons are transferred to oxygen.

Second, the data demonstrated that fumarate also competes with oxygen for the electrons on reduced Frd. Superoxide production in vitro was half-inhibited by 95 µM fumarate and 90% inhibited by about 750 µM. Is there this much fumarate in the re-aerated cell? When anaerobic cells re-enter an aerobic habitat, the influx of oxygen will restore aerobic respiration and, by mass action, the counterclockwise direction of the TCA cycle. The K for fumarate of fumarase A is 600 µM(33) , which, if taken as an indication of the intracellular fumarate level, would by itself indicate that superoxide production by Frd would be largely inhibited. Particularly striking, however, is the fact that both fumarases A and B (the anaerobic isozyme that would be carried over from anaerobic growth) are among the handful of enzymes known to be rapidly inactivated by superoxide(34) . It is plausible that the excess superoxide production by Frd would therefore diminish fumarase activity, forcing an increase in steady-state fumarate concentration and thereby suppressing superoxide production by Frd. Perhaps this effect upon superoxide production favored the retention in this facultative bacterium of superoxide-sensitive fumarases, in contrast to mammalian cells, which have no Frd and express a structurally dissimilar fumarase that is unaffected by superoxide.

Mechanism of Superoxide Production by Frd and Sdh

The single-electron reduction potential of molecular oxygen is -0.16 V; thus it is only likely to be reduced to superoxide by particularly good univalent electron donors. Most biomolecules resist the loss of a single electron and therefore are not sources of superoxide. Fumarate reductase is evidently exceptional, generating superoxide far more rapidly than any other intact metabolic enzyme yet known. Its example demonstrates that metabolic enzymes that are not damaged (unlike xanthine oxidase) and that have not evolved to this purpose (unlike the NADPH oxidase of neutrophils) may nonetheless have the ability to produce large fluxes of superoxide. Frd may provide an opportunity to identify the characteristics that dispose redox enzymes to generate superoxide, potentially facilitating the search for other such enzymes.

Frd, like Sdh, contains a flavin and three iron-sulfur clusters (35) . When acting to reduce fumarate, electrons are passed from reduced menaquinone through the [3Fe-4S] and [2Fe-2S] clusters to the flavin, which then transfers them to fumarate. The experiments with substrate analogues indicated that the flavin, rather than an Fe-S cluster, was the site of electron transfer from Frd to oxygen. Excess succinate had the same effect in Sdh, implying that superoxide evolved from the flavin of that enzyme as well. This localization is consistent with similar observations for xanthine oxidase(36) . The formation of superoxide by a flavin oxidoreductase of E. coli(37) probably reflects the oxidation by molecular oxygen of the reduced flavin product. It may turn out to be generally true that flavoenzymes comprise most of the superoxide sources of physiological significance.

However, it is not true that all flavoenzymes are similarly proficient at generating superoxide. Even excluding oxidases and monooxygenases (38) , the superoxide turnover numbers for dehydrogenase-type flavoproteins vary over 5 orders of magnitude (Table 6). Evidently additional structural or electronic characteristics are critical. The steric accessibility of the isoalloxazine ring may be variable; for example, the exposure of the flavin of xanthine dehydrogenase upon sulfhydryl oxidation is responsible for conferring upon that enzyme its oxidase activity. A second factor may be that fumarate reductase differs from simple flavoproteins, which make little superoxide, in that its redox-active flavin is electronically linked to metal centers. In this respect it resembles xanthine oxidase, another enzyme that is particularly proficient at generating superoxide. The correlation suggests that the secondary redox centers may kinetically promote the univalent electron transfer from the flavin to oxygen. Two mechanisms seem plausible: 1) a flavin-linked metal center might unpair the electrons on the two-electron-reduced enzyme, conferring semiquinone character to the flavin and improving the likelihood of productive orbital overlap with triplet oxygen; or 2) after transfer of the first electron from a dihydroflavin to oxygen, the second might become stably sequestered on a metal center, allowing the nascent superoxide to dissociate from the oxidized flavin. The behavior of xanthine oxidase is most consistent with the second possibility: whereas the fully reduced enzyme quantitatively reduces oxygen divalently to hydrogen peroxide, the two-electron-reduced enzyme transfers a single electron (39, 40) . The residual electron is slow to hop onto oxygen, and it is presumably that delay that enables the previous superoxide product to diffuse away from the flavin without being reduced to peroxide. It will be interesting to see whether fumarate reductase behaves similarly.



Because Sdh has a structure very similar to that of Frd, its lesser tendency to generate superoxide requires additional consideration. One distinction between the two enzymes may lie in the potentials of their flavins relative to those of the iron-sulfur clusters. The cluster potentials of Frd (about -50, -320, and -60 mV for clusters 1 through 3) are significantly lower than those of Sdh (+10, -175, and +65 mV)(35) . The flavin potential of Frd, -55 mV(41) , is well above the -219 mV potential of free flavins and is probably elevated by its covalent linkage to a protein histidine residue(42) . This high potential relative to those of clusters 1 and 3 will push the electron density onto the flavin. In contrast, the higher cluster potentials of Sdh could plausibly tilt the potentials away from the flavin and push electrons onto the metal centers. These arrangements are congruent with the physiological functions of the two enzymes: the electrons of reduced Frd await transfer from the flavin to fumarate, while those of reduced Sdh await transfer from the iron-sulfur clusters to ubiquinone. The collateral effect may be that the high electron density on the flavin accelerates superoxide formation by Frd, while the shift away from that site slows superoxide formation by Sdh. Further work will be necessary to test these ideas.


FOOTNOTES

*
This study was supported by United States Public Health Service Grant GM49640 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Microbiology, University of Illinois, 131 Burrill Hall, 407 South Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-5812; Fax: 217-244-6697; JimImlay{at}qms1.life.uiuc.edu.

(^1)
The abbreviations used are: SOD, superoxide dismutase; Frd, fumarate reductase; Sdh, succinate dehydrogenase; Men, menaquinone; Ubi, ubiquinone; MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

I am grateful to Kay Keyer and Kevin Messner for assistance with some of the experiments reported here and to Gary Cecchini and Bob Gennis for the provision of plasmids. I also thank Irwin Fridovich and Dennis Flint for provocative conversations about this work.


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