Escherichia coli cytochrome c peroxidase is a respiratory oxidase that enables the use of hydrogen peroxide as a terminal electron acceptor Maryam Khademiana and James A. Imlaya,1 aDepartment of Microbiology, University of Illinois, Urbana, IL 61801 Edited by Andreas J. Bäumler, University of California, Davis, CA, and accepted by Editorial Board Member Carl F. Nathan June 3, 2017 (received for review January 28, 2017) Microbial cytochrome c peroxidases (Ccp) have been studied for and the miniferritin Dps protects DNA from Fenton chemistry by 75 years, but their physiological roles are unclear. Ccps are located sequestering loose iron (18–20). When H2O2 levels later fall, glu- in the periplasms of bacteria and the mitochondrial intermem- taredoxins deactivate OxyR by reducing its disulfide bond (21). brane spaces of fungi. In this study, Ccp is demonstrated to be a Data indicate that ∼200 nM steady-state intracellular H2O2 is significant degrader of hydrogen peroxide in anoxic Escherichia sufficient to activate this response (10, 21). Because the cyto- coli ccp . Intriguingly, transcription requires both the presence of plasmic membrane is only semipermeable to H2O2 and the cyto- H2O2 and the absence of O2. Experiments show that Ccp lacks plasm contains robust peroxidase and catalase activities, the internal enough activity to shield the cytoplasm from exogenous H2O2. H2O2 concentration is 5- to 10-fold lower than the external H2O2 However, it receives electrons from the quinone pool, and its flux concentration (10). Accordingly, measurements indicate that rate approximates flow to other anaerobic electron acceptors. In- external H2O2 levels must exceed 2 μM to induce the intracellular deed, Ccp enabled E. coli to grow on a nonfermentable carbon OxyR response (21). Thus, one infers that despite dwelling in Salmonella source when H2O2 was supplied. behaved similarly. mostly hypoxic environments, E. coli must encounter micromolar ccp This role rationalizes repression in oxic environments. We spec- levels of external H2O2 at some stage in its normal lifestyle. ulate that micromolar H2O2 is created both biologically and abiot- Strikingly, even obligate anaerobes manifest H2O2-inducible ically at natural oxic/anoxic interfaces. The OxyR response appears defenses, mediated either by OxyR or by PerR (22–24). The to exploit this H O as a terminal oxidant while simultaneously 2 2 implication is that microbes may confront toxic doses of H2O2 defending the cell against its toxicity. even in low-oxygen environments. Such a conclusion might resolve the conundrum of cytochrome oxidative stress | anaerobic respiration | OxyR c peroxidases. The biochemical activity of these enzymes was first described in 1940 (25), and although their reaction mechanism has he facultative anaerobe Escherichia coli lives adjacent to the been elaborated in detail (26), their physiological role continues to Tepithelial layer of the mammalian gut, where it can respire by perplex. Notably, the bacterial enzymes are expressed only under scavenging trace oxygen that diffuses into the lumen from the anoxic conditions (27, 28). We now report that through YhjA, a epithelial cells. When oxygen levels decline, E. coli can ferment, cytochrome c peroxidase homolog, E. coli can exploit H2O2 as although it is less successful than the coresident obligate an- a terminal oxidant in a form of anaerobic respiration. This rep- aerobes. However, upon excretion, E. coli can thrive, whereas the resents a surprising positive use of H2O2 in metabolism. YhjA is obligate anaerobes enter a period of stasis. induced only when oxygen is scarce and H2O2 is present, implying In fully oxic habitats, E. coli must cope with reactive oxygen that micromolar H2O2 can be found at oxic–anoxic interfaces. We species that are generated internally through the adventitious − oxidation of redox enzymes (1). Superoxide (O2 ) and hydrogen Significance peroxide (H2O2) are potentially toxic, because they oxidatively inactivate cytoplasmic enzymes that use exposed [4Fe–4S] clus- – Hydrogen peroxide has been regarded exclusively as a hazard ters or ferrous iron atoms as prosthetic cofactors (2 6). H2O2 for bacteria; its sources and concentrations in natural habitats also oxidizes the intracellular pool of unincorporated iron are uncertain. The cytochrome c peroxidase of Escherichia coli through the Fenton reaction, thereby generating hydroxyl radi- exhibits an expression pattern and flux rate that provides cals that can damage DNA (7). To protect itself from these oxi- surprising insights into these issues. This periplasmic enzyme is dants, E. coli—like virtually all organisms—routinely synthesizes − induced only when H2O2 is present and molecular oxygen is superoxide dismutases to keep O2 levels low and catalase and absent. Intriguingly, it was ineffective as a defensive enzyme, NADH peroxidase to minimize H2O2. As a consequence, wild- but through its linkage to the quinone pool it did enable E. coli type E. coli grows well in oxic environments. to respire using H2O2 as an anaerobic electron acceptor. We In addition to these basal protections, most bacteria have the suggest that both chemical and biotic processes generate mi- capacity to ramp up their defenses when they encounter extra- cromolar H2O2 at oxic/anoxic interfaces and that this scenario is cellular H2O2 (8, 9). Exogenous H2O2 moves freely through common enough that microbes have evolved strategies to porins into the periplasm, and because it is small and uncharged, productively exploit the H2O2. it can gradually diffuse across the cytoplasmic membrane into the cell interior, where it poses a hazard (10). When H2O2 levels Author contributions: M.K. and J.A.I. designed research; M.K. performed research; M.K. rise in E. coli, the H2O2 directly oxidizes the sensory cysteine and J.A.I. analyzed data; and M.K. and J.A.I. wrote the paper. residue of the OxyR transcription factor, triggering the forma- The authors declare no conflict of interest. tion of a disulfide bond that locks the protein into an activated This article is a PNAS Direct Submission. A.J.B. is a guest editor invited by the Editorial conformation (11, 12). OxyR then activates the expression of Board. defensive genes (13). Catalase and NADH peroxidase activities See Commentary on page 8678. are boosted more than 10-fold. Suf proteins improve the repair 1To whom correspondence should be addressed. Email: [email protected]. – of iron sulfur clusters (14, 15), the manganese importer MntH This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. allows Mn to replace ferrous iron in mononuclear enzymes (16, 17), 1073/pnas.1701587114/-/DCSupplemental. E6922–E6931 | PNAS | Published online July 10, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1701587114 Downloaded by guest on September 24, 2021 suggest that YhjA be renamed Ccp, and we continue to use Ccp PNAS PLUS throughout this paper. Results Cytochrome c Peroxidase Is a Fourth Enzyme That Scavenges H2O2. E. coli has three enzymes that are known to degrade H2O2 in vivo: the KatG and KatE catalases, plus the NADH peroxidase AhpCF. For convenience, mutants that lack all three enzymes − (katG katE ahpCF) are termed hydroperoxidase-deficient (Hpx ) − (29). When Hpx mutants are grown aerobically, they do not clear SEE COMMENTARY micromolar H2O2 from medium at a significant rate (Fig. 1). However, we were surprised to find that when the same strain was grown and exposed to H2O2 under anoxic conditions, it dis- played substantial ability to degrade H2O2 (Fig. 1, rightmost bar). The culture filtrate did not degrade H2O2 (Fig. S1), indicating that the scavenging activity resided within the cells. We considered the possibility that H2O2 might be eliminated through Fenton reactions with the cytoplasmic pool of unincorporated iron. However, our calculations indicated that the cell was unlikely to − − Fig. 2. Ccp scavenges H2O2 in anoxic Hpx cells. The Hpx parent strain and contain enough iron to support the observed rate of H2O2 clear- mutant derivatives were grown anaerobically, and the rate of H2O2 scav- ance (SI Materials and Methods), and indeed the scavenging ac- enging was measured. Cross-hatched bars: The cytochrome c maturation tivity persisted when cells were perfused with dipyridyl, a chelator machinery is required for Ccp activity. Spotted bar, far right: the Hpx− Δccp that fully blocks intracellular Fenton chemistry (7) (Fig. S1). mutant was genetically complemented using pACYC184-ccp under its own Therefore, we focused upon E. coli enzymes that have been promoter. Asterisks represent statistical significance (*P ≤ 0.05; **P ≤ 0.01; − reported to display peroxidase activities in vitro: thiol peroxidase ***P ≤ 0.001; ns, P > 0.05). The single mutants other than Hpx Δccp and − − Hpx Δccm were not significantly different from Hpx . None of the double (Tpx) (30), bacterioferritin comigratory protein (BCP) (31), a − − mutants were significantly different from Hpx Δccp. Strains used: Hpx glutathione peroxidase homolog (BtuE) (32), a predicted cyto- − − − (LC106), Hpx Δbcp (MK150), Hpx Δtpx (MK154), Hpx ΔbtuE (MK158), chrome c peroxidase (YhjA, or Ccp) (28), and osmotically in- − − − − Hpx Δccp (MK146), Hpx Δcyd Δcyo (SSK53), Hpx ΔappBC (MK180), Hpx ducible peroxiredoxin (OsmC) (33). The physiological roles of ΔosmC (MK208), Hpx− Δccp Δbcp (MK172), Hpx− Δccp Δtpx (MK174), Hpx− − − these enzymes are uncertain, and they have not been reported to Δccp ΔbtuE (MK176), Hpx Δccp ΔappBC (MK182), Hpx Δccp Δcyd − − − degrade