(Received for publication, April 6, 1995; and in revised form, June 8, 1995)
From the
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.
Synthesis of superoxide dismutase (SOD(); 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 10M
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` 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 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. 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.
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.
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.
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.
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
).
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 -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.
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.
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 -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 -glycerolphosphate, L-lactate, or NADH (not shown). Table 5shows that those
substrates whose dehydrogenases are most kinetically competent at
menaquinone reduction,
-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.
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
frdABCD) 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.''
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 HO
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
O
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 -glycerolphosphate dehydrogenase via
menaquinone. This route is responsible for the vast majority of O
production during the respiration of
-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 -glycerolphosphate
dehydrogenase to the [3Fe-4S] cluster of Frd. Excess
succinate
[
OOC-CH
-CH
-COO
]
or its analogues, malonate
[
OOC-CH
-COO
]
and malate
[
OOC-CH
-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:
-glyc-P,
-glycerolphosphate; DHAP, dihydroxyacetone phosphate; Glp,
-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.
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.
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
-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.
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.
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.