Endogenous superoxide is a key effector of the sensitivity of a model obligate anaerobe

Zheng Lua,1, Ramakrishnan Sethua,1, and James A. Imlaya,2

aDepartment of Microbiology, University of Illinois, Urbana, IL 61801

Edited by Irwin Fridovich, Duke University Medical Center, Durham, NC, and approved March 1, 2018 (received for review January 3, 2018) It has been unclear whether superoxide and/or hydrogen peroxide fects (3, 4). Thus, these phenotypes confirmed the potential tox- play important roles in the phenomenon of obligate anaerobiosis. icity of reactive oxygen species (ROS), and they broadly supported This question was explored using Bacteroides thetaiotaomicron,a the idea that anaerobes might be poisoned by endogenous major fermentative bacterium in the human gastrointestinal tract. oxidants. Aeration inactivated two families—[4Fe-4S] dehydratases The metabolic defects of the mutant E. coli strains were sub- and nonredox mononuclear —whose homologs, in sequently traced to damage to two types of enzymes: dehy- contrast, remain active in aerobic . Inactivation- dratases that depend upon iron- clusters and nonredox rate measurements of one such enzyme, B. thetaiotaomicron fu- enzymes that employ a single atom of ferrous iron (5–9). In both marase, showed that it is no more intrinsically sensitive to oxi- enzyme families, the metal centers are solvent exposed so that dants than is an E. coli . Indeed, when the E. coli they can directly bind and activate their substrates. Superoxide B. thetaiotaomicron enzymes were expressed in , they no longer and H2O2 are tiny molecules that cannot easily be excluded from could tolerate aeration; conversely, the B. thetaiotaomicron en- active sites, and they have high affinity for iron. The upshot is zymes maintained full activity when expressed in aerobic E. coli. that they directly ligand and oxidize the enzyme metal centers. Thus, the aerobic inactivation of the B. thetaiotaomicron enzymes The oxidized iron atoms dissociate, activity is lost, and the is a feature of their intracellular environment rather than of the pathways fail. B. thetaiotaomicron enzymes themselves. possesses superoxide Superoxide and H2O2 are continuously formed in aerobic cells dismutase and peroxidases, and it can repair damaged enzymes. because molecular oxygen adventitiously oxidizes redox enzymes However, measurements confirmed that the rate of reactive oxy- (10–12). Due to its substantial titers of scavenging enzymes, WT gen species production inside aerated B. thetaiotaomicron is far E. coli can suppress this threat. The question remains as to higher than in E. coli. Analysis of the damaged enzymes recovered whether these ROS poison obligate anaerobes. Among the from aerated B. thetaiotaomicron suggested that they had been whose oxygen sensitivity has received particular atten- inactivated by superoxide rather than by hydrogen peroxide. Ac- tion are members of the Bacteroidetes (13–18). These carbohy- cordingly, overproduction of superoxide dismutase substantially drate fermenters are among the dominant bacteria in the protected the enzymes from aeration. We conclude that when this mammalian gut (19), where they grow alongside E. coli. How- anaerobe encounters oxygen, its internal superoxide levels rise ever, in contrast to E. coli, Bacteroides species quickly stop high enough to inactivate key catabolic and biosynthetic enzymes. growing upon aeration. Notably, they do so despite possessing a Superoxide thus comprises a major element of the oxygen sensi- substantial retinue of SOD, catalase, and peroxidases (16, 20– tivity of this anaerobe. The extent to which molecular oxygen 22). analysis of aerated Bacteroides thetaiotaomicron exerts additional direct effects remains to be determined. Significance oxidative stress | obligate anaerobiosis | Bacteroides | reactive oxygen species Microbes display profound differences in their tolerance for oxygen, and this trait organizes the structure of many micro- he phenomenon of obligate anaerobiosis is the most obvious bial communities. However, the molecular basis of oxygen Tnatural manifestation of oxidative stress. Many microorgan- sensitivity is not well understood. In this study we determined isms can only grow in anoxic places. This restriction is a domi- that Bacteroides thetaiotaomicron, an abundant member of nant factor in the organization of microbial ecosystems in soil the human intestinal flora, is incapacitated by superoxide and gut, where respiring organisms help to shield the majority of stress when it enters a fully oxic environment. The key differ- anaerobes from the encroachment of oxygen. In 1971, McCord ence from oxygen-tolerant bacteria lies not in its defensive et al. (1) published a survey of scavenging enzymes that implied a systems, nor in the nature of the affected enzymes, but in the possible cause of obligate anaerobiosis. In contrast to oxygen- rate of endogenous oxidant formation. Anaerobes thrive in tolerant microbes, the anaerobes that they examined contained oxygen-poor environments because they deploy low-potential little or no superoxide dismutase (SOD) or catalase—which electron-transfer pathways; these results suggest that an an- suggested that, upon aeration, these microbes would be poisoned cillary effect is the reactivity of these pathways with oxygen, − by superoxide (O2 ) or hydrogen peroxide (H2O2). The table thereby generating enough reactive oxygen species to pre- that was published has been widely circulated, and this correla- clude oxic growth. tion is still cited in textbooks as a likely explanation for obligate anaerobiosis. Author contributions: Z.L., R.S., and J.A.I. designed research, performed research, ana- In 1986, Carlioz and Touati (2) performed a key experimental lyzed data, and wrote the paper. test of the idea, by deleting the SOD genes from the facultative The authors declare no conflict of interest. bacterium Escherichia coli. The resultant mutant grew at normal This article is a PNAS Direct Submission. rates in the absence of oxygen, but upon aeration it exhibited a Published under the PNAS license. set of severe biosynthetic and catabolic defects. These included 1Z.L. and R.S. contributed equally to this work. deficiencies in the biosynthesis of eight amino acids plus an in- 2To whom correspondence should be addressed. Email: [email protected]. ability to use TCA-cycle substrates as carbon sources. Analogous This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mutants that lacked catalase and peroxidase were generated 1073/pnas.1800120115/-/DCSupplemental. much later, and these mutants exhibited many of the same de- Published online March 20, 2018.

E3266–E3275 | PNAS | vol. 115 | no. 14 www.pnas.org/cgi/doi/10.1073/pnas.1800120115 Downloaded by guest on September 29, 2021 showed that stoppage of growth occurs concomitant with a loss Results PNAS PLUS of carbohydrate (15). Two enzymes in central me- When B. thetaiotaomicron cultures in rich medium were aerated, tabolism lose activity (Fig. 1): fumarase, a member of the iron- growth stopped after ∼40 min (Fig. 2). The static cells remained sulfur dehydratase family, and pyruvate:ferredoxin oxidoreduc- viable; when anoxia was restored hours later, growth resumed tase (PFOR), a key pyruvate-dissimilating enzyme that passes within minutes. We previously noted that the cessation of growth low-potential electrons toward hydrogen formation and/or NAD was accompanied by a diminution of glucose catabolism and the reduction. The fumarase bottleneck is marked by a cessation of parallel inactivation of fumarase and PFOR, key enzymes in succinate production and an unusual release of lactate. When central (15). Fumarase drew our attention because this injury was bypassed by the addition of exogenous fumarate, this enzyme belongs to the family of [4Fe-4S] dehydratases, some succinate production was restored, but the cell instead which are vulnerable to oxygen species that can oxidize their iron-sulfur clusters (5, 25–27). Assays revealed that two other excreted pyruvate, reflecting PFOR failure. Either block should members of this enzyme family, and isopropylmalate be enough to prohibit fermentative growth. − , also progressively lost activity when B. thetaiotaomicron In this study we tested whether O or H O might be in- 2 2 2 was aerated (Fig. 3). volved. Our immediate focus was drawn to fumarase, because its The other family known to be vulnerable to these oxidants vulnerability to ROS is well understood (5, 23, 24). We found comprises enzymes that use solvent-exposed ferrous iron atoms that aeration simultaneously inactivated other iron-sulfur dehy- to catalyze nonredox reactions (6, 7, 9). When E. coli is stripped dratases and mononuclear iron enzymes. These failures were not of its scavenging enzymes, both superoxide and H2O2 can oxidize due to any special sensitivity of the B. thetaiotaomicron enzymes, enzymic Fe(II) cofactors, triggering iron release, the loss of ac- which maintained activity when expressed in aerobic E. coli. tivity, and collapse of the processes to which these enzymes Instead, the cellular environment of aerated B. thetaiotaomicron contribute. We examined two such enzymes in B. thetaiotaomicron: is much more oxidizing than that of E. coli due to a much higher ribulose-5-phosphate 3-epimerase (Rpe) and peptide deformylase rate of endogenous ROS formation. Finally, analysis indicated (Pdf). Both enzymes employ ferrous iron (rather than other di- − that O2 is the specific culprit. These results validate the original valent metals) in B. thetaiotaomicron (Figs. S1 and S2), and both − lost activity when cells were aerated (Fig. 3). suggestion of McCord et al. (1) that O2 toxicity might underlie key aspects of obligate anaerobiosis. The failure of these enzymes in aerated B. thetaiotaomicron stands in sharp contrast to aerobic E. coli, where such enzymes retain full activity in vigorously aerated cultures. We considered the possibility that oxygen-tolerant bacteria have evolved en- zymes that are intrinsically less reactive with oxidants. Fumarases were purified from both B. thetaiotaomicron and E. coli, and their iron-sulfur clusters were reconstituted in anoxic buffers. In vitro, these enzymes can be inactivated by superoxide, hydrogen per- oxide, and molecular oxygen itself. As shown in Fig. 4, these species inactivated the two fumarases with similar rate constants. This result indicated that aspects of the cell environment, rather than of the enzymes themselves, are determinant in whether they retain activity in oxic environments. To test this notion more directly, the fumarase and Rpe ho- mologs were each expressed in both bacteria. To do so, mutants were created that lacked the native enzymes. Fig. 5 A and B shows that both fumarase enzymes remained fully active in E. coli when protein synthesis was blocked and erstwhile anoxic cells were aerated. In contrast, both enzymes lost activity when the same experiment was performed using B. thetaiotaomicron. This pattern was replicated with the Rpe homologs (Fig. 5 C and D). Thus, some aspect of B. thetaiotaomicron is not conducive to the function of oxidant-sensitive enzymes. Direct assays showed that anoxic B. thetaiotaomicron possesses 25% as much SOD activity as does E. coli (28). This difference is not a sufficient explanation for the inactivation of the enzymes, since fumarase and other iron-sulfur enzymes retain activity inside E. coli when its SOD titers are diminished to this level (29). B. thetaiotaomicron also has a catalase and three peroxidases that are devotedtoscavengingH2O2 (22). It is difficult to quantify the in- ternal scavenging activities of peroxidases, but B. thetaiotaomicron and E. coli are similarly effective at clearing H2O2 from laboratory

Fig. 1. Relevant pathways in B. thetaiotaomicron metabolism. Shown in MICROBIOLOGY brackets are enzymes that lose activity when cells are transferred to oxic cultures. Therefore, it is not obvious that enzymes in aerated B. conditions. Fumarase is critical for the redox-balancing branch of central thetaiotaomicron are damaged due to a particular deficiency of metabolism, while PFOR initiates the energy-conserving dissimilation of scavenging systems. Further, we observed that B. thetaiotaomicron pyruvate. Lower-flux pathways are represented by dashed lines. Rpe is is able to repair damaged enzymes. After aerated cells were needed for oxidative flux through the pentose-phosphate pathway; IPMI is returned to anoxic conditions, enzyme activities returned, even necessary for leucine synthesis, and aconitase serves a biosynthetic role in the generation of the α-ketoglutarate family of amino acids. Not depicted: when protein synthesis was blocked (Fig. S3). In sum, although Pdf is essential for the maturation of nascent polypeptides. Acn, aconitase; these two bacteria exhibit some qualitative and quantitative Fum, fumarase; αKG, alpha-ketoglutarate; PEP, phosphoenolpyruvate; OAA, differences in cellular defenses, the differences seem unlikely to . explain the disparity in enzyme fate.

Lu et al. PNAS | vol. 115 | no. 14 | E3267 Downloaded by guest on September 29, 2021 responsible for this enzyme damage in vivo. We examined B. thetaiotaomicron mutant strains that lack either SOD or cata- lase/peroxidases. Fumarase lost activity more rapidly in either mutant than in WT cells, confirming the ability of these oxidants to inactivate the enzyme in vivo and showing that they are partially shielded by these scavenging enzymes in WT cells (Fig. 8). Fumarase inactivation was especially rapid in the SOD mutant. The same pattern was reproduced when Rpe activity was tracked. Strikingly, therateofRpedamageinSOD-deficientB. thetaiotaomicron far exceeded its rate of damage in SOD-deficient E. coli, consistent with our expectation that superoxide is formed more quickly in aerated B. thetaiotaomicron. When superoxide oxidizes the iron cofactors of mononuclear enzymes, the dissociation of iron leaves an apoprotein whose activity can be rapidly restored by the binding of another Fe(II) atom (8). In SOD-deficient E. coli, this process of iron oxidation and rebinding can occur repeatedly, but each cycle provides some chance that a competing metal like Zn(II) will bind in place of Fe(II); this outcome diminishes activity, because in such enzymes zinc is a poor catalyst (8, 9). Interestingly, we observed the same phenomenon in SOD-deficient B. thetaiotaomicron (Fig. 8C). Activity could be restored to the lysates only by adding penicillamine, a good zinc chelator, to extract a blocking metal Fig. 2. Growth ceases upon aeration and resumes when oxygen is removed. from the before the addition of Fe(II). We infer that Cultures growing in anoxic BHIS medium were aerated at the first arrow. At the metal was zinc rather than manganese, because the latter the second arrow, cells were centrifuged and then resuspended in anoxic metal would have furnished substantial activity (Fig. S1) and BHIS. would have dissociated relatively quickly without a chelator (Materials and Methods). B. thetaiotaomicron lacks the two known bacterial manganese importers, MntH and MntABC. When H O is formed inside mutant strains that lack H O - 2 2 2 2 The outcome is different when E. coli Rpe is oxidized by H O scavenging enzymes, the H O diffuses out into the medium, 2 2 2 2 (6). In the latter case, the reaction between the active-site Fe(II) allowing the rate of endogenous formation to be quantified (30). and H O generates a ferryl/hydroxyl species that has a finite Our previous work indicated that H O is formed much more 2 2 2 2 chance of irreversibly damaging the polypeptide. The enzymes quickly in aerated B. thetaiotaomicron than in aerated E. coli: that are oxidized in this way cannot be reactivated. This behavior upon aeration, the OxyR H O stress response was immediately 2 2 was replicated in B. thetaiotaomicron: whereas the Rpe that lost activated only in the obligate anaerobe, and mutants lacking this activity in SOD mutants could be fully reactivated, the enzymes response died quickly (22, 31). The H O measurements were 2 2 that lost activity in catalase/peroxidase mutants could not be repeated under the condition of the present experiments. Anoxically grown cells were washed and resuspended into buffer, to avoid chemical H2O2 production by medium components, and then aerated. When glucose was provided as a carbon source, the rate of endogenous H2O2 formation was 10 times higher in B. thetaiotaomicron than in E. coli (Fig. 6A). When glucose was omitted, the rates were diminished in both strains, suggesting that catabolism is the source of ROS. Notably, the withdrawal of glucose simultaneously slowed the rates at which B. thetaiotao- micron enzymes were damaged (Fig. 6 B and C). We do not have a good way to quantify superoxide formation inside cells. However, studies with E. coli enzymes indicate that both species evolve from the same event, the autoxidation of flavoenzymes (32). Any superoxide is subsequently converted to H2O2, either by SOD or by spontaneous dismutation. Therefore, it seems likely, although not definitive, that the high rate of H2O2 production in B. thetaiotaomicron connotes a similarly high rate of superoxide production. The rate of endogenous H2O2 formation depends upon oxy- gen concentration, in accordance with the model that ROS are primarily formed by the adventitious oxidation of redox enzymes (31). When B. thetaiotaomicron was exposed to a range of oxygen concentrations, the amount of residual fumarase activity depended inversely upon the oxygen level (Fig. 7). This outcome Fig. 3. Iron-sulfur dehydratases and mononuclear Fe(II) enzymes lose ac- is notable in that it suggests that the bacterium may retain sub- tivity when B. thetaiotaomicron is aerated. Top three panels: Three [4Fe-4S] stantial enzyme and pathway functions when oxygen is present at dehydratases. Bottom two panels: Two nonredox Fe(II) enzymes. Chloram- phenicol was added to block new protein synthesis, and cells were aerated in lower levels. glucose buffer. Specific protocols are described in Materials and Methods.In These data, plus the observation that B. thetaiotaomicron this and other figures, error bars represent the SEM from at least three enzymes remain active in aerobic E. coli, indicate that super- measurements. By the final time point, P < 0.001 for inactivation of all en- oxide or H2O2—rather than molecular oxygen—is likely to be zymes shown.

E3268 | www.pnas.org/cgi/doi/10.1073/pnas.1800120115 Lu et al. Downloaded by guest on September 29, 2021 when McCord et al. (1) conducted their survey. It is implicit that PNAS PLUS all microbes occasionally confront enough oxygen to warrant the presence of defensive enzymes.

Why Hasn’t B. thetaiotaomicron Solved Its Oxygen Problem? An important question has been whether the appearance of scav- enging enzymes eliminated the threat of endogenous superoxide and H2O2, or whether these endogenous ROS are still sub- stantial enough to exert toxic effects. The results here suggest that E. coli suffers little enzyme damage from the ROS that it generates, but B. thetaiotaomicron can still be overwhelmed. The difference lies in the amount of ROS that the two bacteria generate. Why doesn’t B. thetaiotaomicron compensate by making higher titers of scavenging enzymes? Two ideas stand out. First, the levels of scavenging enzymes in E. coli already make these among the most abundant proteins in the cell. Even so, aerobic E. coli sits close to the verge of oxidative collapse: calculations suggest that its [4Fe-4S] dehydratases are oxidized and must be repaired every half hour or so (32). Indeed, just a fivefold increase in superoxide level is enough to reduce the steady-state activities of its [4Fe-4S] dehydratases by half (Fig. S4) (29). Since aerated B. thetaiotaomicron produces ROS at 10 times the rate of E. coli,it follows that it would require 10-fold higher titers of scavenging Fig. 4. The B. thetaiotaomicron fumarase is no more sensitive to oxidants in enzymes than E. coli to achieve the same level of enzyme sta- vitro than is the E. coli enzyme. Inactivation rate constants were determined bility. Such an investment of resources, including metal cofactors for the purified enzymes as described in Materials and Methods. Note the to activate the SOD, may be untenable. multipliers indicated above the bar graphs. No significant differences be- Second, B. thetaiotaomicron features key enzymes that may be tween the two enzymes were indicated for any of the three comparisons − damaged by molecular oxygen per se. If oxygen can damage (P = 0.16 for O2, 0.5 for H2O2, and 0.06 for O2 , with the B. thetaiotaomicron enzyme possibly exhibiting less sensitivity). B. theta, B. thetaiotaomicron; enzymes directly, then the value of scavenging enzymes would be inactiv’n, inactivation. limited. B. thetaiotaomicron possesses two pyruvate dissimilating enzymes: pyruvate:formate (Pfl) and PFOR. Pfl is oxygen sensitive by virtue of its glycyl-radical chemistry, which is (Fig. 9). It is important then that when WT cells were aerated, the inactivated Rpe could be fully reactivated by penicillamine/ Fe(II) treatment. The enzyme profile resembled Rpe that was recovered from SOD mutants that had been exposed to oxygen for 30 min. The implication is that superoxide, rather than H2O2, is the primary oxidant that disables these enzymes when B. the- taiotaomicron is aerated. To test this conclusion more directly, SOD was overproduced ∼15-fold in WT B. thetaiotaomicron. When these cells were aerated, both fumarase and Rpe were substantially protected (Fig. 10). Some damage still occurred, due either to the residual superoxide or to H2O2 itself, and growth did not resume (see Discussion). However, this outcome demonstrates that superox- ide is the oxidant that damages these enzymes when this obligate anaerobe encounters oxygen. Discussion Life evolved in an anoxic world. Ferrous iron was readily avail- able (33), and its proficiency as a surface and electron-transfer catalyst resulted in its recruitment into many enzymes. The emerging metabolic pathways became configured around the chemistry that iron can catalyze. Two billion years later, the appearance of oxygenic photosys- tem II put organisms at risk, because oxygen oxidizes redox en- zymes to make ROS, and the ROS can then poison the exposed iron cofactors of enzymes. It was once believed that microor- MICROBIOLOGY ganisms responded either by evolving SOD and catalase or by retreating to anoxic habitats. However, as the facility of lateral Fig. 5. Fumarase and Rpe enzymes from both bacteria retain activity in gene transfer became apparent, it seemed unlikely that bacteria aerated E. coli but not in aerated B. thetaiotaomicron.(A and B) Fumarases B would be trapped in anoxic environments simply for lack of from E. coli and B. thetaiotaomicron were expressed in anoxic E. coli (A)orB. thetaiotaomicron (B). Chloramphenicol was added to block new protein scavenging enzymes. Indeed, further investigations revealed that synthesis, and activity was tracked after cells were aerated. (C and D) even anaerobes maintain a cohort of scavenging enzymes: if not Analogous experiments were performed with Rpe from both sources SODs and catalases, then superoxide reductases and peroxidases (34, expressed in E. coli (C)orB. thetaiotaomicron (D). Strains and protocols are 35). The latter enzymes were either unknown or underappreciated detailed in Materials and Methods. B. theta, B. thetaiotaomicron. *P < 0.05.

Lu et al. PNAS | vol. 115 | no. 14 | E3269 Downloaded by guest on September 29, 2021 Fig. 6. The rate of enzyme damage correlates with the rate of endogenous ROS formation. (A) The efflux of endogenous H2O2 was tracked after aeration of − Hpx derivatives of B. thetaiotaomicron (ΔkatE ΔahpC, Δrbr1, Δrbr2; circles) and E. coli (ΔkatG, ΔkatE, ΔahpCF; squares). The cells were grown in BHIS (B. thetaiotaomicron)orLB(E. coli) and then washed and aerated in buffer containing chloramphenicol and either no carbon source (open symbols) or 0.2%

for continued catabolism. No significant H2O2 accumulated in sterile buffer. (B and C) Under the conditions of A, the activities of Rpe and Pdf were monitored in B. thetaiotaomicron. Statistical analyses compare values at each time point between glucose-fed and -starved cells. *P < 0.05; **P < 0.01. B. theta, B. thetaiotaomicron; FU, fluorescence units; glc, glucose.

quenched by direct reaction with oxygen, a radical itself (36). (31). Those data did not support the idea that the respiratory PFOR may be the primary route of pyruvate consumption, and chain was involved in high-rate ROS production. when cells are aerated its activity diminishes on a time frame Moving forward, there are some hints. The rate of H2O2 for- similar to that of fumarase (15). The inactivation mechanism is mation in aerated B. thetaiotaomicron is so high that it exceeds not known, but it seems likely to involve overoxidation of its low- fluxes through most biosynthetic pathways. We there- potential redox clusters (37). Either oxygen or ROS derived from fore suspect that the ROS may evolve from the PFOR- it similarly inactivates the enzyme in vitro. Oxygen sensitivity in ferredoxin-/RNF (ferrodoxin:NAD ) vitro is not enough to predict that oxygen will also poison PFOR route of central metabolism. After PFOR passes electrons to in vivo: S-adenosylmethionine radical enzymes (38), for example, ferredoxin, the reduced ferredoxin delivers them either to a hy- are oxygen-sensitive in vitro but function normally inside aerobic drogenase or to RNF; NADH generated by the latter then cells. However, if molecular oxygen itself does directly inactivate proceeds to NADH dehydrogenase of the respiratory chain (43). PFOR in B. thetaiotaomicron, then the failure of this bacterium The redox moieties in these enzymes carry electrons at low po- to make higher levels of scavenging enzymes can be justified: tentials, making transfer to oxygen energetically favorable. Fur- elevated oxygen concentrations would inactivate PFOR, and thus ther, this is among the few metabolic pathways of B. block central metabolism, regardless of the titers of scavengers. thetaiotaomicron that E. coli does not share, which could explain This possibility was noted in the original report of McCord et al. the discrepancy in ROS rate. The gradual slowing of H2O2 re- (1). The mechanism is under investigation. Direct PFOR in- lease after aeration (Fig. 6A) plausibly reflects the progressive activation would also explain the failure of SOD overproduction inactivation of PFOR. This hypothesis awaits experimental to enable aerobic growth—although that outcome might also evidence. arise from the inactivation of ribonucleotide reductase. B. the- In sum, this study shows that both classes of enzymes known to taiotaomicron relies upon an oxygen-sensitive glycyl-radical ri- be damaged by ROS in nonscavenging mutants of E. coli are also bonucleotide reductase whose presence in B. thetaiotaomicron presumably reflects its commitment to its anoxic habitat.

What Is the Source of the Toxic ROS? Our data show that the rapid formation of superoxide is the particular feature that condemns ROS-sensitive enzymes to inactivity when B. thetaiotaomicron is aerated. We do not know the origin of all of this ROS. Oxygen has a triplet electronic structure that constrains it to accept electrons in univalent steps (39), and so the univalent redox properties of flavoenzymes and quinones have prompted workers to focus upon respiratory chains as plausible sites of ROS pro- duction. Surprisingly, genetic studies have indicated that this chain is, at best, a minor contributor to ROS formation in aer- obically grown E. coli (11). However, ROS formation in E. coli rises immediately after the aeration of anoxically grown cells, and in this specific circumstance the source was identified as the anaerobically induced respiratory enzyme fumarate reductase (12, 40). The redox flux through this flavoprotein is even greater in B. thetaiotaomicron than in E. coli, and so we conjectured that it might be the main source of ROS stress. In the related bac- terium, Bacteroides fragilis, mutants that lack fumarate reductase were subsequently observed to generate lower amounts of H2O2 (41); interpretation was not straightforward, however, since Fig. 7. Fumarase activity in B. thetaiotaomicron depends upon the oxygen these mutants grow much more slowly than do WT cells (42). A concentration. Fumarase activity was measured after 1 h of exposure to the recent biochemical analysis showed that the B. thetaiotaomicron indicated oxygen concentration. Full aeration is represented by 22% oxygen. fumarate reductase has a distinct electronic structure that pre- The dotted line represents prior data (31) showing that the rate of endog-

cludes reactions with oxygen, unlike that of its E. coli homolog enous H2O2 production is proportionate to oxygen concentration.

E3270 | www.pnas.org/cgi/doi/10.1073/pnas.1800120115 Lu et al. Downloaded by guest on September 29, 2021 PNAS PLUS

Fig. 8. Scavenging enzymes slow the rate of enzyme damage in aerated B. thetaiotaomicron.(A and B) Fumarase (A) or Rpe (B) activity was monitored after − − the aeration of B. thetaiotaomicron strains that lacked enzymes that scavenge H2O2 (Hpx ) or superoxide (SOD ). B also depicts the rate of Rpe inactivation upon the aeration of E. coli SOD− strains. Inactivation is much faster in the B. thetaiotaomicron strain, suggesting that superoxide levels are significantly higher. P values in A compare mutant to WT activities; P values in B compare the activities of SOD− B. thetaiotaomicron with those of SOD− E. coli.(C) Extracts − were prepared from the B. thetaiotaomicron SOD strain at the indicated time points of aeration. Rpe activities were assayed before (gray bars) or after (black bars) treatment with Fe(II) alone or penicillamine and then Fe(II) (hatched bars). The ability to reactivate with Fe(II) alone defines the fraction of Rpe in an apoprotein form, whereas the increasing requirement for preliminary penicillamine treatment indicates that Zn(II) increasingly occupied the enzyme metal- . The pattern matches that observed in superoxide-stressed E. coli (8). [As-extracted enzyme activity was not altered by either Fe(II) or penicil- lamine/Fe(II) treatment.] *P < 0.05; **P < 0.01; ***P < 0.001.

damaged by simple aeration of WT B. thetaiotaomicron. The were from Sigma. His Gravitrap was obtained from GE Healthcare. D-Glucose, · origin of the ROS and its involvement in the inactivation of Hepes, Tris HCl, and MgCl2 were purchased from Fisher. Glycylglycine was from PFOR are the next problems to solve. Acros Organics. Amplex Ultrared reagent was purchased from Invitrogen through Thermo Fisher Scientific. Materials and Methods Cell Media and Growth. BHI-supplemented medium (BHIS; ref. 44) contained Chemicals. Brain-heart infusion (BHI) broth was purchased from Difco. Hemin −1 37 gL of BHI broth, 15 μM hemin chloride, 4 mM L-cysteine hydrochloride, hydrochloride, L-cysteine, L-cystine, fumaric acid, ferrous ammonium sulfate hexahydrate, DTT, maltose, β-lactose, 2,2′-dipyridyl, citraconate, disodium and 22.6 mM sodium bicarbonate. Antibiotics were supplemented into the medium when required as 20 μgmL−1 erythromycin, 200 μgmL−1 genta- L-malic acid, DL-trisodium isocitrate, horse heart c, xanthine, μ −1 μ −1 ′ bovine xanthine oxidase, bovine liver catalase, E. coli iron-containing SOD, micin, 15 gmL chloramphenicol, and 200 gmL 5 -fluorodeoxyuridine. – imidazole, antibiotics (ampicillin, erythromycin, gentamicin, and chloram- Luria Bertani (LB) medium contained 10 g tryptone, 10 g NaCl, and 5 g phenicol), 5′-fluorodeoxyuridine, HRP, α-glycerophosphate dehydrogenase/ extract per liter. Defined medium was composed of minimal A salts (45) μ triosephosphate isomerase from rabbit muscle, manganese(II) chloride tet- supplemented with 0.2% casein acid hydrolysate, 0.2% glucose, 5 g/mL rahydrate, cobalt(II) chloride hexahydrate, zinc(II) sulfate heptahydrate, thiamine, 0.02% MgSO4 heptahydrate, and 0.5 mM tryptophan. Maltose ferric(III) chloride, EDTA, D-penicillamine, D-ribose 5-phosphate disodium (0.5%) and 0.2% lactose were added as a carbon sources where indicated. salt, D-ribulose 5-phosphate disodium salt, D-xylulose 5-phosphate sodium The BHIS, LB, and defined media were adjusted to pH 7.0 before + salt, NADH, NAD , formate dehydrogenase, formyl-Met-Ala-Ser tripeptide, sterilization. thiamine pyrophosphate, isopropylthiogalactopyranoside (IPTG), 2,6- Anoxic growth was performed in a Coy anaerobic chamber that contained

pyridinedicarboxylic acid, Tris (2-carboxyethyl)phosphine (TCEP), and 30% H2O2 an atmosphere of nitrogen (85%), hydrogen (10%), and (5%). MICROBIOLOGY

Fig. 9. The reactivatibility of Rpe recovered from aerated cells suggests that superoxide rather than H2O2 is the damaging species. Rpe was recovered after − − 3 h of aeration of Hpx , SOD , or WT cells. Left shows that the H2O2-damaged enzyme can only be partially reactivated; Middle shows that superoxide- damaged enzyme can be fully reactivated after penicillamine extracts the competing metal; and Right shows that Rpe from WT cells can be fully reactivated, − consistent with damage by O2 rather than H2O2.*P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.

Lu et al. PNAS | vol. 115 | no. 14 | E3271 Downloaded by guest on September 29, 2021 Fig. 10. Overproduction of SOD protects B. thetaiotaomicron enzymes from the aeration. (A) SOD was overexpressed 15-fold from a plasmid (psod). (B and C) Activities of fumarase and Rpe after aeration of strains containing the vector or SOD-overproducing plasmid. BHIS-grown cells were washed and then aerated in buffer containing glucose. **P < 0.01; ***P < 0.001.

Autoclaved media were immediately transferred to the anaerobic chamber 20 mM fumarate, 200 μg/mL gentamicin, and 20 μg/mL chloramphenicol. In and stored for ≥24 h to ensure the outgassing of residual oxygen. Buffers experiments using these strains, maltose (0.4% wt/vol) was added to BHIS used in this study were kept in an anaerobic chamber for ≥1 wk before use. medium to activate the SusA promoter. All experiments were conducted upon exponential-phase cultures that had To express fumarases in E. coli,afumA fumB fumC mutant strain (JH400; doubled at least four times subsequent to dilution of overnight cultures. ref. 49) lacking all three native E. coli fumarases was used as the host. Genes

Typically, cells were inoculated to 0.005 OD600 into warm growth medium, encoding the E. coli fumA and B. thetaiotaomicron fum were cloned behind and they were grown under anoxic conditions to 0.10–0.20 OD600 before the lac promoter of the low-copy number vector pWKS30 by HindIII/BamHI. biochemical or physiological measurements were begun. The RBS of E. coli gapA was inserted upstream of each gene. Sequences of The impact of oxygen exposure and removal upon cell growth was ob- primers for PCR are listed in Table S3. Construction was confirmed by di- served by culturing WT B. thetaiotaomicron in anoxic BHIS medium to an gestion. To enable expression, 0.2% lactose replaced glucose in the defined

OD600 of 0.1. Cultures were then transferred out of the anaerobic chamber medium. and aerated with vigorous shaking at 37 °C for ∼3 h. The cells were then The E. coli rpe gene was amplified by PCR, digested with BamHI/SacI, and

centrifuged, resuspended in an equal volume of prewarmed anoxic BHIS inserted into pNLY-PsusA plasmid. The RBS (21 bp) was included at the up- media, and cultured once again in the anaerobic chamber at 37 °C. Growth stream region of the gene as pNLY-PsusA plasmid lacks it. The pNLY-PsusA-rpe was monitored by OD600. plasmidwastransformedintotheparentB. thetaiotaomicron rpe mutant strain using a biparental mating procedure, and selected for gentamicin Gene Deletions. The bacterial strains and plasmids that were used in this study (200 μg/mL) and chloramphenicol (20 μg/mL) resistance. Expression was are listed in Tables S1 and S2. The BT5482 Δtdk strain served as the parent achieved by growing the recombinant plasmid containing the B. thetaio- strain for construction of deletion mutants using a published method (46). taomicron strain in BHIS medium supplemented with 0.5% maltose in place (The tdk gene encodes thymidine kinase, an enzyme in the salvage pathway of glucose. of pyrimidine biosynthesis, and it is nonessential under the conditions of The B. thetaiotaomicron rpe gene was inserted into pWKS30 (50) and these experiments.) The B. thetaiotaomicron rpe (BT_3946) and sod transformed into an E. coli strain that lacks the rpe gene. The gene was (BT_0655) genes were identified by BLAST analysis using E. coli rpe and cloned behind the lac promoter by HindIII/BamHI. The RBS of the gapA gene sodB sequences as queries. The consequent mutations were confirmed by was again included upstream of the B. thetaiotaomicron rpe gene. The genome PCR and enzyme assays. Note that under standard assay conditions plasmid was transformed into the E. coli Δrpe strain. Expression was B. thetaiotaomicron rpe mutants retained a few percent of the activity of achieved by replacing glucose with lactose in defined minimal A medium. WT cells; such activity has been observed in other bacteria as well (47) and is Inactivation of the heterologous Rpe enzymes was compared with in- due to the promiscuity of other enzymes, which themselves lack iron and are activation of the native enzymes in WT strains. not sensitive to oxidants. The E. coli rpe deletion mutation was obtained from theKeiocollection(48)attheE. coli Genetic Stock Center. The mutation was Enzyme Purifications. The E. coli and B. thetaiotaomicron fumarase coding transferred to recipient strains by P1 transduction (45), and inheritance was regions were inserted into the pET16b vector (Novagen), which was trans- again verified by PCR analysis and enzyme assay. Genomic DNA was isolated formed to BL21 (DE3). IPTG (0.5 mM) was added to 1 L log-phase culture

from both E. coli and B. thetaiotaomicron using DNeasy blood and tissue kit near 0.5 OD600 to induce the protein expression. After 3 h at 37 °C, cells were (Qiagen), as instructed by the manufacturer. harvested by centrifugation. Fumarase was purified under aerobic condi- Deletion of the sole fum gene (BT_2256) of B. thetaiotaomicron did not tions at 4 °C following the standard Ni-NTA resin purification protocol succeed until we included 20 mM fumarate in the growth media. Exogenous (Novagen). Aliquots of the purified protein were stored at −80 °C and fumarate can be imported and used as a substrate for fumarate reductase, reactivated before use. thereby restoring to fum mutants the redox-balancing and energetic func- To reactivate the purified protein, the 1-mL anaerobic reaction system

tions of the succinate-production pathway. contained 1–5 μM fumarase, 0.5 mM Fe(NH4)2(SO4)2, 2.5 mM DTT, 2.8 mM cysteine, and 0.1 μMpurifiedE. coli IscS protein, in 50 mM NaPi buffer (pH 7.2) Overproduction of Enzymes. The SOD of B. thetaiotaomicron was over- (51). The reaction was incubated at room temperature in the anaerobic produced by cloning the native gene behind its own promoter onto a chamber for ≥2 h, and then dipyridyl was added to a final concentration of multicopy plasmid. The sod (BT_0655) coding sequence, plus 520 bp imme- 1 mM to stop the process. After 5 min, the reaction was filled to 4 mL by NaPi diately upstream that contains the promoter region and ribosome binding buffer and concentrated from 4 mL to 0.1 mL three times by 10-kDa Millipore 2+ site, were amplified by PCR and inserted into pNLY-PsusA vector (22) by filtering tubes. This step removed the extra Fe and DTT. All steps were BamHI/SacI. The clone was screened on anaerobic BHIS plates (200 μg/mL performed under anoxic conditions. Reactivated enzyme was then used gentamicin and 20 μg/mL chloramphenicol). Overproduction of SOD in without further storage. standard BHIS medium was confirmed by assay. Rpe from B. thetaiotaomicron was purified following the protocol used To express E. coli and B. thetaiotaomicron fumarases in the B. thetaio- for its E. coli homolog (6). The rpe gene was cloned using pET16b vector, and taomicron fum mutant strain, the coding sequences of E. coli fumB and B. the resulting pRpe-His10 was overexpressed in the E. coli BL21(DE3) strain. thetaiotaomicron fum were amplified by PCR; the ribosome binding site The Rpe-His10 protein was then purified using His Gravitrap. The His10 tag (RBS) of B. thetaiotaomicron fum (21 bp) was added upstream of each gene. was removed after purification. Purified protein was >95% pure as indicated Primers are listed in Table S3. Each DNA fragment was inserted into the by SDS/PAGE analysis. The enzyme was stored at 4 °C.

pNLY-PsusA vector by BamHI/SacI, behind the SusA promoter. Construc- We found that E. coli transketolase obtained from commercial sources tion was confirmed by digestion. The plasmid was conjugated into the was contaminated with Rpe; therefore, E. coli transketolase A was purified B. thetaiotaomicron fum mutant and selected in BHIS medium containing under aerobic conditions as described in ref. 52. Cell extracts were prepared

E3272 | www.pnas.org/cgi/doi/10.1073/pnas.1800120115 Lu et al. Downloaded by guest on September 29, 2021 from LB culture of E. coli BL21 Δrpe strain containing the tktA over- medium, while allowing the intracellular ROS formation that occurs as a by- PNAS PLUS expression plasmid pET16b-tktA. The enzyme was >95% pure as indicated product of . by SDS/PAGE analysis. The purified enzyme was stored at −80 °C in 50 mM Fumarase inactivation was also examined upon aeration of an E. coli fumA Tris·HCl, pH 8, 20% glycerol. fumB fumC mutant strain (JH400) that was complemented with either E. coli The E. coli enzyme IscS enables the in vitro reconstruction of [4Fe-4S] fumarase A or B. thetaiotaomicron fumarase expressed from plasmids. Cul- clusters on apoprotein forms of fumarase and other enzymes. IscS was pu- tures were grown in minimal A medium in which lactose replaced glucose as rified as described (51). a carbon source, enabling expression of fum genes from the lac promoter. Ampicillin (50 μg/mL) was included to maintain the expression plasmid. As Enzyme Assays. For assays of oxidant-sensitive enzymes, cells were first with the B. thetaiotaomicron experiments, log-phase anaerobic cultures centrifuged and then washed in anoxic buffers, and cell extracts were then were harvested, resuspended in aerobic buffer with lactose and chloram- prepared by sonication in anoxic buffers in an anaerobic chamber. For fu- phenicol, and shaken under room air at 37 °C. At time points, cultures were marase assays, washed cells were sometimes frozen and stored at −80 °C; for harvested aerobically and moved back to the chamber for preparation of IMPI and aconitase assays, cells were lysed and assayed immediately. All cells extracts and measurement of enzyme activity. A parallel anaerobic assay reactions were assembled in the anaerobic chamber in a sealed cuvette culture was harvested as an oxygen-free control. before being moved to a laboratory spectrophotometer. Reactions were at A similar approach was taken to track fumarase activities in cells exposed room temperature (RT). Fumarase activity was determined in 50 mM sodium to defined concentrations of oxygen. Log-phase cultures in BHIS were washed phosphate (pH 7.3) containing 50 mM L-malate; production of fumarate was and suspended in NaPi buffer (pH 7.2, 0.2% glucose plus chloramphenicol) monitored at 250 nm (53). Cell densities were adjusted so lysates contained that was either anaerobic or that had been steadily gassed for 1 h with a ∼1 mg/mL protein. Isopropylmalate isomerase (IPMI) activity was measured selected ratio of nitrogen and air. The gassing ratio was established by mixing by monitoring the decrease in citraconate absorbance at 235 nm in 100 mM gas flow from nitrogen and air cylinders at a Y-intersection; the gas stream Tris-Cl (pH 7.6) (54). The aconitase assay was performed in 100 mM Tris-Cl (pH was then bubbled through a water trap (to ensure hydration) and finally 8.0) containing 20 mM DL-trisodium isocitrate; the formation of aconitate was through the sample tube (12). Parafilm was stretched over the tube to measured at 240 nm (55). SOD activities were determined by the xanthine/ minimize exposure to laboratory air, which was excluded anyway because of xanthine oxidase method, under oxic conditions (56). the positive in-to-out pressure. Cultures were injected into the tube through the parafilm with a Hamilton syringe. At intervals, cultures were harvested Rpe converts ribulose-5-phosphate to xylulose-5-phosphate, which was anaerobically to prepare cell extracts and to measure enzyme activities. then detected in an enzyme-coupled assay through NADH oxidation as a decrease in A (57). Rpe and Pdf were assayed immediately upon har- To monitor the activities of Rpe and Pdf, log-phase E. coli and B. the- 340 ∼ vesting, without storage. The Rpe assay from crude extracts was performed taiotaomicron cells were grown at least four generations to 0.2 OD in anoxic LB and BHIS media, respectively. Cells were then washed twice and in 50 mM glycylglycine buffer (pH 8.5) at RT. Extracts were prepared in the resuspended in anoxic 50 mM glycylglycine buffer (pH = 8.5) containing anaerobic chamber. Cells were washed twice with ice-cold 50 mM glycyl- 100 μg/mL chloramphenicol, either in the absence and presence of 0.2% glycine buffer containing 1 mM EDTA, pH 8.5, and resuspended in 1 mL of glucose, as indicated. Cultures were either incubated in the anaerobic the same buffer without EDTA. Samples were then sonicated for 2 min (3 s chamber or were transferred to an external laboratory shaker. At intervals, on/3 s off), and lysates were cleared by centrifugation at 20,000 × g for cultures were returned to the chamber, extracts were prepared, and 3 min. Assays were performed within 5 min of cell lysis to avoid metal dis- enzymes were assayed. To test the ability of cells to reactivate Rpe, the air- sociation. A typical assay (500 μL) contained 50 mM glycylglycine buffer exposed cells were returned to the anaerobic chamber, washed once with (pH 8.5), 1 mM ribulose 5-phosphate, 1 mM ribose 5-phosphate, 1 unit of same anoxic buffer, resuspended in anaerobic BHIS buffer containing transketolase, 0.2 mM MgCl , 2 mM thiamine pyrophosphate, 5 mM EDTA, 2 chloramphenicol (100 μg/mL), and incubated anaerobically for 1 h at 37 °C. 1 unit of α-glycerophosphate dehydrogenase, 10 units of triosephosphate Cell extracts were then prepared for assay. isomerase, and 0.2 mM NADH. Pdf removes the formyl group from the model peptide formyl-Met-Ala- In Vivo Repair of B. thetaiotaomicron Fumarase. The ability of intact cells to Ser. The released formate is then oxidized to CO and H O by formate de- 2 2 repair oxidatively damaged fumarase was tested. WT B. thetaiotaomicron hydrogenase, with reduction of NAD+, and NADH formation is monitored by cells were aerated for 2 h in 50 mM NaPi (pH 7.2) buffer containing 0.2% the increase in A (58). Cells were centrifuged in the anaerobic chamber at 340 glucose and 100 μg/mL chloramphenicol to inactivate fumarase. The cul- 4 °C in 50 mM Hepes buffer with 25 mM NaCl (pH 7.5) plus 1 mM EDTA, ture was returned to the chamber. An aliquot was removed and prepared for washed twice in the same buffer, and finally resuspended in 1 mL of the assay. The remaining cells were centrifuged and then resuspended in anoxic same buffer without EDTA. Samples were then sonicated for 2 min (3 s on/3 s 37 °C BHIS medium containing chloramphenicol. The culture was subsequently off), and lysates were cleared by centrifugation at 12,000 rpm for 3 min. harvested and lysed, and fumarase activity was measured after 2–12 h. Assays were performed within 5 min of cell lysis to avoid metal dissociation. A typical assay (500 μL) contained 50 mM Hepes buffer with 25 mM NaCl + Analysis of Rpe and Pdf Metallation Status. One cannot use the metal content (pH 7.5), 10 mM NAD , 1 unit of formate dehydrogenase, 1 mM formyl-Met- of purified mononuclear enzymes as an indicator of their native metallation Ala-Ser, and cell extracts, all under anoxic conditions at RT. state in vivo, because this enzyme family readily exchanges metals with the Transketolase was assayed by standard methods (52) in a reaction mix- buffer during the purification process. Instead, the presence of iron in the ture containing 50 mM glycylglycine buffer, pH 8.5; 5 mM DTPA; 1 unit of active site can be deduced by susceptibility to H O immediately after extract α 2 2 -glycerophosphate dehydrogenase; 10 units of triosephosphate isomer- preparation (7). To test whether as-extracted Rpe and Pdf contained iron, ase; 1 mM xylulose 5-phosphate; 1 mM ribose 5-phosphate; and 0.2 mM fresh cell extracts from E. coli or B. thetaiotaomicron were treated with NADH in a final reaction volume of 500 μL. Absorbance was monitored 100 μMH2O2 at 25 °C for 10 min (8). Catalase (30 U/mL) was added to stop at 340 nm. the reaction. Rpe assays were performed before and after H2O2 treatment. To reactivate the enzymes in vitro, 90 μLH2O2-challenged cell extracts were Measurement of Enzyme Activities After Aeration. To track the effect of incubated with 500 μM (final concentration) of the desired metal, and en- aeration upon native cellular fumarase, aconitase, and IPMI enzyme activities, zyme activity was remeasured. Where indicated, Pdf was preincubated for a 150-mL culture of B. thetaiotaomicron was grown in anaerobic BHIS me- 10 min with 500 μM TCEP before the addition of the metal. dium from 0.01 to 0.25 OD600 and then centrifuged, washed once, and The activity and stability of Rpe metallated with Fe, Zn, and Mn were also resuspended in 10 mL RT anoxic 50 mM NaPi buffer (pH 7.2). Bacteria were measured. Rpe was recovered after purification in its Zn(II)-Rpe form; Zn grown similarly when expressing fumarases from a plasmid, except that

often binds to mononuclear enzymes during purification. Zinc was removed MICROBIOLOGY 100 μg/mL chloramphenicol was included. Five-milliliter aliquots of the sus- from the purified protein by chelation using dipicolinic acid (20 mM) and pended anaerobic bacteria were added to 25 mL oxic or anoxic NaPi buffer, EDTA (100 μM) at 25 °C for 1 h. The apoenzyme was then diluted 1,000-fold each containing glucose and enough chloramphenicol to block protein into anoxic buffer containing 100 μM of the test metal at 25 °C. Activity was synthesis (100 μg/mL chloramphenicol for strains without vector, 500 μg/mL then measured. To test whether differentially metallated enzyme was sen-

chloramphenicol for strains with vector pNLY-SusA). The anoxic suspension sitive to H2O2, the enzyme was incubated with 100 μMH2O2 for another was incubated at 37 °C in the anaerobic chamber, while the oxic suspension 5 min. The reaction was terminated by the addition of catalase (30 U/mL), was transferred to an outside incubator and shaken vigorously. At time and the enzyme was reassayed. In separate experiments, the spontaneous dis- points, cultures were transferred back to chamber, and cell extracts were sociation rate of the metals was determined by incubating metal-loaded enzyme prepared and assayed. The aeration of cultures was conducted in buffered in the presence of 1 mM EDTA at RT and tracking activity. Iron-, manganese-, and

glucose to avoid the chemical production of H2O2 by components of BHIS zinc-loaded Rpe displayed dissociation half-times of 60 min, 5 min, and >8h,

Lu et al. PNAS | vol. 115 | no. 14 | E3273 Downloaded by guest on September 29, 2021 respectively, in accord with intrinsic metal softness and in close agreement with Determination of Inactivation Rate Constants. To measure the rate constants

the values determined for the E. coli enzyme (6). Rpe recovered in extracts from with which H2O2 inactivates fumarases (5), 90 nM or 180 nM H2O2 was used anoxic cells lost activity in the presence of EDTA with a half-time of 60 min, to inactivate the purified/reconstituted enzymes. Reactions were per- matching that of iron-cofactored enzyme. formed with 10 nM enzyme at RT in 1 mL 50 mM NaPi in the anaerobic In some experiments, the metallation status of Rpe was tested after the chamber; enzyme activities were measured at intervals. Rate constants − − aeration of WT, Hpx ,orSOD cells. Cell extracts were prepared and assayed were calculated. The reconstitution protocol had removed unincorporated under anoxic conditions. An aliquot of extract was then metallated with iron, which was necessary to avoid chemical H2O2 degradation through the ferrous iron (1 mM) for 10 min and then reassayed; any boost in activity Fenton reaction. represents apoprotein in the original sample. Separate aliquots were tested − For the measurement of inactivation rate constants of O2 , a competition for mismetallated Rpe; such enzyme is poorly active but can be reactivated if assay system was employed in vitro (27). In 1 mL 50 mM KPi, 2 nM B. the- the inappropriate metal is extracted from the active site. To chelate metal − taiotaomicron fumarase or 18 nM E. coli fumarase was exposed to O2 from the active site of Rpe, cell extracts were incubated with 0.5 mM EDTA produced by xanthine oxidase in the presence of 0, 0.37, 1.48, or 3.7 nM for 3 h or with 0.5 mM penicillamine for 1 h under anoxic conditions at 25 °C iron-containing SOD (Sigma). Xanthine was 50 μM, and xanthine oxidase was (8). Rpe activity was completely absent after chelation. To remetallate the 5 mU/mL. Catalase (200 U/mL) was included in the reaction mix to prevent enzymes, the desired metals were added to a final concentration of 1 mM, enzyme damage by H O . The reaction components were mixed in the an- and the mixture was incubated anaerobically for 10 min at 25 °C. Activity 2 2 aerobic chamber in a tube, which was then transferred out of the chamber was then remeasured. Collectively, these procedures distinguished enzyme and aerated by pipetting the reaction mixture down the side of the tube. At that was correctly metallated from apoprotein, mismetallated protein, and intervals, excess SOD was added to stop the reaction, and the remaining activity irreversibly damaged protein. − 9 −1 −1 was determined by assay. SOD reacts with O2 at a rate of 2 × 10 M s .By comparing the inactivation rates of fumarase in the presence and absence of Measurement of H2O2 Production by Aerated Cells. Mutants that lack catalase − − SOD, the inactivation rate constants of O can be calculated (27). and peroxidase activities are denoted as hydroperoxidase-deficient, or Hpx 2 [katG katE ahpCF for E. coli (3) and ΔkatE ΔahpC Δrbr1 Δrbr2 for B. the- To measure the inactivation rate constants for molecular O2,purified and reactivated 10–50 nM E. coli or B. thetaiotaomicron fumarases were taiotaomicron (22)]. These cells cannot scavenge endogenous H2O2 and re- lease it directly into the medium. Cultures were grown for more than four added at RT to anoxic NaPi buffer containing 500 U/mL of both SOD and catalase. The buffer was then saturated with air (to achieve 220 μMO )by generations in LB (E. coli)orBHIS(B. thetaiotaomicron)toanOD600 = 0.2. Cells 2 were then centrifuged and washed twice with anoxic 50 mM glycylglycine pipetting along the cuvette wall aerobically. The incubation extended for

buffer, pH 8.5, at RT. The cell pellets were then resuspended to OD600 = 0.2 in 20 min. Excess catalase and SOD (both 500 U/mL) were included in all as- anoxic 50 mM glycylglycine buffer, pH 8.5, supplemented with or without says. Samples were returned at intervals to the chamber and assayed un- 0.2% glucose, and exposed to air with vigorous shaking at 37 °C. At 3-min der anoxic conditions. Activity was stable when enzymes were maintained intervals, aliquots were removed and centrifuged, and supernatants were under anoxic conditions. stored on dry ice/ethanol until sample collection was complete. Supernatants

were then thawed and immediately assayed for H2O2 concentration using ACKNOWLEDGMENTS. This work was supported by Grant GM049640 from Amplex Red/HRP (59). Standard curves were performed in the same media. the NIH.

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