Title: Role of a Bacterial Organic Hydroperoxide Detoxification System in Preventing Catalase Inactivation
Abstract: In the gastric pathogen Helicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host. Here we report a connection unidentified previously between the two stress resistance enzymes. We observed that the catalase in ahpC mutant cells in comparison with the parent strain is inactivated partially (approximately 50%). The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy. To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides on H. pylori catalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase. H. pylori catalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H2 O2. However, it inactivated is by lower concentrations of organic hydroperoxides (the substrate of AhpC). Treatment with a molar ratio of 1:400 t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50%. UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species. To further support the idea that organic hydroperoxides, which accumulate in the ahpC mutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found in ahpC mutant cells with that found in wild type cells. The results showed that the total amount of extractable lipid hydroperoxides in the ahpC mutant cells is approximately three times that in the wild type cells. Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation. In the gastric pathogen Helicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host. Here we report a connection unidentified previously between the two stress resistance enzymes. We observed that the catalase in ahpC mutant cells in comparison with the parent strain is inactivated partially (approximately 50%). The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy. To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides on H. pylori catalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase. H. pylori catalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H2 O2. However, it inactivated is by lower concentrations of organic hydroperoxides (the substrate of AhpC). Treatment with a molar ratio of 1:400 t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50%. UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species. To further support the idea that organic hydroperoxides, which accumulate in the ahpC mutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found in ahpC mutant cells with that found in wild type cells. The results showed that the total amount of extractable lipid hydroperoxides in the ahpC mutant cells is approximately three times that in the wild type cells. Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation. The ability of pathogenic bacteria to resist oxidative stress is crucial to their infectiousness and pathogenesis in the host (1Storz G. Zheng M. Storz G. Hengge-Aronis R. Bacterial Stress Responses. ASM Press, Washington, D. C.2000: 47-60Google Scholar, 2Miller R.A. Britigan B.E. Clin. Microbiol. Rev. 1997; 10: 1-18Crossref PubMed Google Scholar). To defend against reactive oxygen species that can cause protein oxidation, lipid peroxidation, and DNA damage, living organisms rely on serial enzymatic machinery. Superoxide dismutase, catalase, and alkyl hydroperoxide reductase (AhpC) 1The abbreviations used are: AhpC, alkyl hydroperoxide reductase; t-BOOH, t-butyl hydroperoxide. are virtually ubiquitous enzymes that confer oxidative stress resistance (3Imlay J.A. Linn S. Science. 1988; 240: 1302-1309Crossref PubMed Scopus (1670) Google Scholar, 4Fridovich I. J. Biol. Chem. 1997; 272: 18515-18517Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 5Lowen P. Scandalois J.G. Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Press, Plainview, NY1997: 273-308Google Scholar, 6Netto L.E.S. Chae H.Z. Kang S.W. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1996; 271: 15315-15321Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Superoxide dismutase dismutates the superoxide anion into hydrogen peroxide and molecular oxygen, and catalase breaks down H2O2 into water and O2. AhpC reduces organic hydroperoxides (ROOH, also extended to include HOOH) into the corresponding non-toxic alcohol (ROH). The elimination of organic hydroperoxides is particularly important for living cells because organic hydroperoxides can initiate a lipid peroxidation chain reaction and consequently propagate free radicals, leading to DNA and membrane damage (7Halliwell B. Gutteridge J.M. Biochem. J. 1984; 219: 1-14Crossref PubMed Scopus (4577) Google Scholar). AhpC is a component of a large family of thiol-specific antioxidant proteins, with roles that are not generally well understood (8Chae H.Z. Robison K. Poole L.B. Church G Storz G Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (706) Google Scholar, 9Wood Z.A. Schroder E. Harris J.R. Poole L.B. Trends Biochem. Sci. 2003; 28: 32-40Abstract Full Text Full Text PDF PubMed Scopus (2136) Google Scholar). A highly successful human bacterial pathogen Helicobacter pylori induces a strong inflammatory response within the host, thereby releasing a high level of host-derived toxic oxygen species, but H. pylori can survive and colonize persistently in the harsh conditions of the gastric mucosa (10Bagchi D. Bhattachatya G. Stohs S.J. Free Radic. Res. 1996; 24: 439-450Crossref PubMed Scopus (138) Google Scholar, 11Hazell S.L. Harris A.G. Trend M.A. Mobley H.L.T. Mendz G.L. Hazell S.L. Helicobacter pylori: Physiology and Genetics. ASM Press, Washington, D. C.2001: 167-175Google Scholar, 12Ramarao N. Gray-Owen S.D. Meyer T.F. Mol. Microbiol. 2000; 38: 103-113Crossref PubMed Scopus (95) Google Scholar). To account for this capability, H. pylori possesses superoxide dismutase, KatA, and AhpC enzymes (11Hazell S.L. Harris A.G. Trend M.A. Mobley H.L.T. Mendz G.L. Hazell S.L. Helicobacter pylori: Physiology and Genetics. ASM Press, Washington, D. C.2001: 167-175Google Scholar). In addition, some other factors have been identified that play important roles in oxidative stress resistance. NapA, a ferritin-like iron-binding protein, is involved in oxidative stress resistance probably through sequestering free iron in the cells (13Olczak A.A. Olson J.W. Maier R.J. J. Bacteriol. 2002; 184: 3186-3193Crossref PubMed Scopus (92) Google Scholar, 14Cooksley C. Jenks P.J. Green A. Cockayne A. Logan R.P. Hardie K.R. J. Med. Microbiol. 2003; 52: 461-469Crossref PubMed Scopus (97) Google Scholar); also, a NADPH quinone reductase (MdaB) confers oxidative stress resistance by maintaining the quinone pool of the cell in the reduced state (15Wang G. Maier R.J. Infect. Immun. 2004; 72: 1391-1396Crossref PubMed Scopus (101) Google Scholar). The disruption of each individual gene for superoxide dismutase, KatA, AhpC, or MdaB affects severely the ability of the bacterium to colonize the host stomach (15Wang G. Maier R.J. Infect. Immun. 2004; 72: 1391-1396Crossref PubMed Scopus (101) Google Scholar, 16Harris A.G. Wilson J.E. Danon S.J. Dixon M.F. Donegan K. Hazell S.L. Microbiology. 2003; 149: 665-672Crossref PubMed Scopus (95) Google Scholar, 17Olczak A.A. Seyler R.W. Olson J.W. Maier R.J. Infect. Immun. 2003; 71: 580-583Crossref PubMed Scopus (77) Google Scholar, 18Seyler R.W. Olson J.W. Maier R.J. Infect. Immun. 2001; 69: 4034-4040Crossref PubMed Scopus (132) Google Scholar), demonstrating the importance of these enzymes in oxidative stress resistance and host colonization. H. pylori expresses abundant levels of catalase and AhpC proteins (19Jungblut P.R. Bumann D. Haas G. Zimny-Arndt U. Holland P. Lamer S. Siejak F. Aebischer A. Meyer T.F. Mol. Microbiol. 2000; 36: 710-725Crossref PubMed Scopus (246) Google Scholar). The genetic and biochemical characterization of H. pylori catalase and AhpC has been performed in different laboratories (13Olczak A.A. Olson J.W. Maier R.J. J. Bacteriol. 2002; 184: 3186-3193Crossref PubMed Scopus (92) Google Scholar, 20Hazell S.L. Evans D.J. Graham D.Y. J. Gen. Microbiol. 1991; 137: 57-61Crossref PubMed Scopus (99) Google Scholar, 21Lundstrom A.M. Bolin I. Microb. Pathog. 2000; 29: 257-266Crossref PubMed Scopus (34) Google Scholar, 22Baker L.M. Raudonikiene A. Hoffman P.S. Poole L.B. J. Bacteriol. 2001; 183: 1961-1973Crossref PubMed Scopus (162) Google Scholar, 23Odenbreit S. Wieland B. Haas R. J. Bacteriol. 1996; 178: 6960-6967Crossref PubMed Google Scholar, 24Harris A.G. Hinds F.E. Beckhouse A.G. Kolesniow T. Hazell S.L. Microbiology. 2002; 148: 3813-3825Crossref PubMed Scopus (69) Google Scholar). H. pylori catalase is a homotetrameric protein in which each subunit has a molecular mass of 59 kDa. It is a monofunctional catalase without peroxidase activity. A unique property of H. pylori catalase is that it has a pI of >9. Another property of H. pylori catalase, which is distinct from other typical catalases, is its stability at very high concentrations of H2O2 (20Hazell S.L. Evans D.J. Graham D.Y. J. Gen. Microbiol. 1991; 137: 57-61Crossref PubMed Scopus (99) Google Scholar). H. pylori cells were shown to be resistant to a high concentration (∼100 mm) of H2O2, and this resistance was abolished in katA– mutants (24Harris A.G. Hinds F.E. Beckhouse A.G. Kolesniow T. Hazell S.L. Microbiology. 2002; 148: 3813-3825Crossref PubMed Scopus (69) Google Scholar). H. pylori AhpC is a major component of the AhpC-thioredoxin-thioredoxin reductase-dependent peroxiredoxin system that catalyzes the reduction of hydroperoxides including H2O2 and organic hydroperoxides (22Baker L.M. Raudonikiene A. Hoffman P.S. Poole L.B. J. Bacteriol. 2001; 183: 1961-1973Crossref PubMed Scopus (162) Google Scholar, 25Windle H.J. Fox A. Eidhin D.N. Kelleher D. J. Biol. Chem. 2000; 275: 5081-5089Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), as well as the reduction of peroxynitrite (26Bryk R. Griffin P. Nathan C. Nature. 2000; 407: 211-215Crossref PubMed Scopus (571) Google Scholar). It was extremely difficult to obtain an ahpC knock-out mutant (21Lundstrom A.M. Bolin I. Microb. Pathog. 2000; 29: 257-266Crossref PubMed Scopus (34) Google Scholar, 22Baker L.M. Raudonikiene A. Hoffman P.S. Poole L.B. J. Bacteriol. 2001; 183: 1961-1973Crossref PubMed Scopus (162) Google Scholar), but the mutant was obtained eventually by screening transformants at a very low O2 (1% partial pressure) condition (13Olczak A.A. Olson J.W. Maier R.J. J. Bacteriol. 2002; 184: 3186-3193Crossref PubMed Scopus (92) Google Scholar). AhpC mutant cells were shown to exhibit severe growth sensitivity to hydroperoxides and to the superoxide-generating agent, paraquat (13Olczak A.A. Olson J.W. Maier R.J. J. Bacteriol. 2002; 184: 3186-3193Crossref PubMed Scopus (92) Google Scholar). We studied the relationship between these two important antioxidant proteins and discovered that the organic hydroperoxide reductase (AhpC) plays a role that was unidentified previously in protecting catalase from inactivation by organic hydroperoxides. For the first time, it is shown that the loss of AhpC function leads to a significant increase of organic hydroperoxides within the cells and that these hydroperoxides are potent inhibitors of catalase. H. pylori Strains and Growth Conditions—H. pylori strain ATCC43504 or the isogenic mutants were cultured on Brucella agar (Difco) plates (called BA plates) supplemented with 5% fetal bovine serum. Cultures of H. pylori were grown microaerobically at 37 °C in an incubator containing 5% CO2 and 2% oxygen. Chloramphenicol (50 μg/ml) or kanamycin (40 μg/ml) was added in the medium for culturing mutants. DNA Techniques—All DNA manipulations were performed as described previously (27Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982Google Scholar). Chromosomal DNA was extracted from H. pylori with the Aquapure genomic DNA extraction kit (Bio-Rad). Plasmid DNA preparations were carried out with the QiaPrep Spin mini kit (Qiagen). DNA fragments or PCR products were purified from agarose gels with the Qiaquick gel extraction kit (Qiagen). PCR was performed in a 2400 thermal cycler (PerkinElmer Life Sciences) with Taq or Pfu DNA polymerase (Fisher). Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA). Construction of H. pylori Mutants—To construct a katA mutant, a 953-bp fragment containing the H. pylori katA gene with genomic DNA from strain ATCC43504 as a template was amplified by PCR using primers katAF (5′-TCCATAAGAGAACAAGCGCC-3′) and katAR (5′-CAACAATGTGATTACGGCCG-3′). The PCR fragment was cloned directly into the pGEM-T vector (Promega), according to the manufacturer's instruction, to generate pGEM-katA. The host strain used for cloning was Escherichia coli DH5α. Subsequently, a chloramphenicol acetyl transferase cassette was inserted at the unique HindIII site within the katA sequence of pGEM-katA. The recombinant plasmid was then introduced into H. pylori by natural transformation via allelic exchange, and chloramphenicol-resistant colonies were isolated. The disruption of the gene in the genome of the mutant strain (ATCC43504, katA:Cm) was confirmed by PCR showing an increase in the expected size of the PCR product. With a similar procedure, the ferritin mutant strain (ATCC43504, pfr:Kan) was constructed as follows. A 1213-bp fragment containing the H. pylori pfr gene was amplified by PCR using primers pfrF (5′-TGGCTAGTTTTAAGGGCATG-3′) and pfrR (5′-AAGCGCAAAATTTGCAAGCG-3′) and cloned into the pGEM-T vector. Subsequently, the 301-bp HindIII fragment within the pfr gene was replaced by a kanamycin resistance cassette. The construction of other mutant strains (sodB, mdaB, ahpC1, ahpC2, ahpC,napA, and napA) in our laboratory was described previously (13Olczak A.A. Olson J.W. Maier R.J. J. Bacteriol. 2002; 184: 3186-3193Crossref PubMed Scopus (92) Google Scholar, 15Wang G. Maier R.J. Infect. Immun. 2004; 72: 1391-1396Crossref PubMed Scopus (101) Google Scholar, 18Seyler R.W. Olson J.W. Maier R.J. Infect. Immun. 2001; 69: 4034-4040Crossref PubMed Scopus (132) Google Scholar). An H. pylori ATCC43504 katA,ahpC double mutant was constructed in this study by transforming ahpC:Kan (type I) mutant strain with the plasmid pGEM-katA:Cm. Cell-free Extract and Membrane Fraction—Plate-grown H. pylori cells were harvested and suspended in phosphate-buffered saline. The cells were collected by centrifugation (10,000 × g for 10 min), resuspended in phosphate-buffered saline, and broken by two passages through a French pressure cell at 18,000 pounds/in2. Crude extracts were then cleared of unbroken cells by centrifugation at 10,000 × g for 10 min. The supernatant (cell-free extract) was then subjected to ultracentrifugation (45,000 × g for 60 min) to obtain the membrane fraction (the pellet). Protein Concentration Determination and Gel Electrophoresis—Protein concentrations were determined with a bicinchoninic acid protein assay kit (Pierce). For SDS-PAGE, 5 μg of cell extract was placed into an SDS buffer, boiled for 5 min, and applied to a denaturing 12.5% acrylamide gel. Densitometric measurements were made for all the protein bands on the SDS gel, and the portion (percentage) attributed to a specific protein (KatA or AhpC) was calculated. Based on this percentage, the molecular weight of the protein, and the total protein concentration, we estimated the molar concentration of KatA or AhpC in the cell extract. Purification of Catalase—Native H. pylori catalase was purified following a similar method described by Radcliff et al. (28Radcliff F.J. Hazell S.L. Kolesnikow T. Doidge C. Lee A. Infect. Immun. 1997; 65: 4668-4674Crossref PubMed Google Scholar). Briefly, H. pylori cells grown on plates were harvested by suspension in 0.1 m sodium phosphate buffer (pH 7.5). After centrifugation, the cell pellet was resuspended in the buffer. Cells were disrupted by three cycles through a French pressure cell at 18,000 pounds/in2, and the lysate was centrifuged at 28,000 × g for 10 min to remove the cell debris. The supernatant was then collected and subjected to ultracentrifugation at 100,000 × g for 45 min. The supernatant was applied to a SP-Sepharose cation exchange column (Amersham Biosciences) that had been equilibrated with 25 mm sodium phosphate buffer (pH 7.5), and proteins were eluted by the creation of a gradient with 1 m NaCl in 25 mm sodium phosphate buffer (pH 7.5). Catalase-positive fractions were selected by checking for oxygen-reducing activity in 3% H2O2. The pooled catalase-positive fractions were further purified by gel filtration chromatography using a Sephacryl S-200 column (Amersham Biosciences) and eluted with 25 mm sodium phosphate buffer (pH 7.5). The purified catalase was then filtered to sterilize and stored at 4 °C, protected from light. Determination of Catalase Activity—The quantitative catalase activity of H. pylori cell extract or purified catalase protein was determined following the method described by Hazell et al. (20Hazell S.L. Evans D.J. Graham D.Y. J. Gen. Microbiol. 1991; 137: 57-61Crossref PubMed Scopus (99) Google Scholar). Briefly, catalase activity was measured spectrophotometrically at 25 °C by following the decrease in absorbance at 240 nm (ϵ240 nm = 43.48 m–1 cm–1) of 13 mm H2O2 in phosphate-buffered saline. All assays were repeated to give 12 rate determinations for the first minute of reaction. One unit was defined as the amount of enzyme that catalyzes the oxidation of 1 μmol of H2 O2– min 1 under the assay condition. Determination of Lipid Hydroperoxides—The amount of lipid hydroperoxides within H. pylori cell extracts or the membrane fractions was determined with a lipid hydroperoxide assay kit (Cayman Chemical, Ann Arbor, MI) following the manufacturer's instruction. Briefly, lipid hydroperoxides within a sample were first extracted into chloroform, which eliminates any interference caused by hydrogen peroxide or endogenous ferric ions in the sample. The lipid hydroperoxides in the extracted sample were used directly in the assay by reacting them with ferrous ions. The resulting ferric ions were detected using thiocyanate ions as the chromogen by measuring absorbance at 500 nm (ϵ500nm = 16,667 m–1 cm–1). An ethanolic solution of 13-hydroperoxyoctadecadienoic acid was used as a lipid hydroperoxide standard. Determination of Organic Peroxide Reductase Activity in Cell Extract—t-Butyl hydroperoxide (t-BOOH) was added to the H. pylori wild type cell extract to a final concentration of 1 mm. At various time points, an aliquot of the sample was removed and assayed for the remaining amount of t-BOOH using the lipid hydroperoxide assay kit as described above. The organic peroxide reductase activity was expressed as the decrease in the amount (nmol) of t-BOOH/min/mg of total protein in the cell extract. EPR Spectroscopy—In this study, EPR spectroscopy was applied to whole cells, cell extracts, or purified catalase to monitor changes of the catalase heme environment. For whole cell samples, a 5-ml cell suspension in phosphate-buffered saline (A600nm = 8) was incubated with 20 mm des-ferrioxamine at 37 °C for 15 min. The cells were then centrifuged, washed with cold 20 mm Tris-HCl (pH 7.4), resuspended in a final volume of 0.4 ml of the same buffer, and frozen in 3-mm quartz EPR tubes by immersion in liquid nitrogen. For cell extracts or purified catalase, the samples were incubated (for 30 min at room temperature) with different concentrations of hydroperoxides, as indicated, and frozen immediately in EPR tubes. Samples were stored at –78 °C for EPR spectroscopic analysis. X band (∼9.6 GHz) EPR spectra were recorded on a Bruker ESP-300E EPR spectrometer equipped with an ER-4116 dual mode cavity and an ESR-9 flow cryostat (Oxford Instruments). The intensity of the EPR signals was normalized to the OD of whole cell samples and the protein absorption band of cell-free extracts and purified samples. Monitoring KatA and AhpC Proteins in H. pylori Cell Extract by SDS-PAGE—Both KatA and AhpC are major proteins expressed in H. pylori cells. Hazell et al. (20Hazell S.L. Evans D.J. Graham D.Y. J. Gen. Microbiol. 1991; 137: 57-61Crossref PubMed Scopus (99) Google Scholar) reported that catalase accounts for ∼1% of the total protein of the cell in H. pylori. The proteome analysis of Jungblut et al. (19Jungblut P.R. Bumann D. Haas G. Zimny-Arndt U. Holland P. Lamer S. Siejak F. Aebischer A. Meyer T.F. Mol. Microbiol. 2000; 36: 710-725Crossref PubMed Scopus (246) Google Scholar) showed that AhpC (also called TsaA) is the third most abundant protein in H. pylori. The expression level of these proteins in H. pylori cells is visualized easily on SDS-PAGE. We identified the specific protein bands by (a) the expected molecular weight, (b) the loss of the band in the corresponding mutant strain, and (c) direct N-terminal sequencing of the protein band. The regulation mechanisms for the expression of KatA and AhpC in H. pylori are currently unclear, but they may involve regulation at both transcriptional and post-translational levels. Using SDS-PAGE, we measured the net expression level of the proteins. There are some variations in the protein expression levels by different strains (data not shown). The profiles of the total proteins in the parent strain ATCC43504 and the isogenic mutant strains katA, ahpC, or napA are shown in Fig. 1. Compared with the wild type strain, the corresponding protein band of KatA, AhpC, and NapA was missing in the respective mutant strains. As shown previously (13Olczak A.A. Olson J.W. Maier R.J. J. Bacteriol. 2002; 184: 3186-3193Crossref PubMed Scopus (92) Google Scholar), there are two types of ahpC mutants, with the type I mutant overexpressing NapA. Based on densitometric measurement of the protein bands on the gel, KatA and AhpC each constitute 2–3% of the total proteins in the wild type cell. Compared with other bacteria, for example Campylobacter jejuni (29Baillon M.L. van Vliet A.H. Ketley J.M. Constantinidou C. Penn C.W. J. Bacteriol. 1999; 181: 4798-4804Crossref PubMed Google Scholar), the expression levels of KatA and AhpC are much higher in H. pylori. Decreased Catalase Activity in ahpC Mutant Cells—H2O2 is a general agent of oxidative stress to the cells. To determine the relative contributions of H. pylori superoxide dismutase, KatA, AhpC, NapA, or MdaB in resistance to H2O2, we determined the H2O2 decomposing activity of each mutant strain compared with the wild type strain. As shown in Fig. 2A, wild type H. pylori exhibited a high level of catalase activity (∼3000 units/mg of total protein), whereas, knock-out mutants in katA (the catalase gene) resulted in a complete loss of catalase activity. As expected, the disruption of other antioxidant genes such as sodB (the gene for superoxide dismutase) or mdaB (the gene for NADPH quinone reductase) did not affect significantly catalase activity of the cells. To our surprise, however, the catalase activity in gene-targeted ahpC mutant cells was determined to be approximately half of that in the wild type cells (Fig. 2A). It was observed in Pseudomonas aeruginosa that catalase activity is affected by the mutation of a separate gene (30Ma J.F. Ochsner U.A. Klotz M.G. Nanayakkara V.K. Howell M.L. Johnson Z. Posey J.E. Vasil M.L. Monaco J.J. Hassett D.J. J. Bacteriol. 1999; 181: 3730-3742Crossref PubMed Google Scholar); a bfrA mutant of P. aeruginosa had only 47% the KatA activity of wild type strain, despite possessing the wild type expression level of KatA. BfrA, composed of 24 subunits and capable of binding 700 iron atoms, is the major iron storage protein in P. aeruginosa (31Moore G.R. Kadir F.H. al-Massad F.K. Le Brun N.E. Thomson A.J. Greenwood C. Keen J.N. Findlay J.B. Biochem. J. 1994; 304: 493-497Crossref PubMed Scopus (32) Google Scholar). The results of Ma et al. (30Ma J.F. Ochsner U.A. Klotz M.G. Nanayakkara V.K. Howell M.L. Johnson Z. Posey J.E. Vasil M.L. Monaco J.J. Hassett D.J. J. Bacteriol. 1999; 181: 3730-3742Crossref PubMed Google Scholar) suggest that BfrA is required as a source of iron for the heme prothetic group of KatA. H. pylori possess two iron storage proteins, NapA and Pfr. NapA is a homologue of bacterial DNA-protecting proteins, and its molecular structure has been determined (32Tonello F. Dundon W.G. Satin B. Molinari M. Tognon G. Grandi G. Del Giudice G. Rappuoli R. Montecucco C. Mol. Microbiol. 1999; 34: 238-246Crossref PubMed Scopus (147) Google Scholar, 33Zanotti G. Papinutto E. Dundon W. Battistutta R. Seveso M. Giudice G. Rappuoli R. Montecucco C. J. Mol. Biol. 2002; 323: 125-130Crossref PubMed Scopus (128) Google Scholar). It has a dodecameric structure (12 subunits) that is capable of binding up to 500 iron atoms (32Tonello F. Dundon W.G. Satin B. Molinari M. Tognon G. Grandi G. Del Giudice G. Rappuoli R. Montecucco C. Mol. Microbiol. 1999; 34: 238-246Crossref PubMed Scopus (147) Google Scholar). Pfr is the major iron storage protein in H. pylori (34Bereswill S. Waidner U. Odenbreit S. Lichte F. Fassbinder F. Bode G. Kist M. Microbiology. 1998; 144: 2505-2516Crossref PubMed Scopus (69) Google Scholar). As a typical ferritin, Pfr forms a 24-mer structure and binds more than 2000 iron atoms (35Andrews S.C. Adv. Microb. Physiol. 1998; 40: 281-351Crossref PubMed Google Scholar). As shown in Fig. 2A, H. pylori KatA activity was not affected significantly by the loss of either NapA or Pfr. Therefore, the observation that H. pylori KatA activity is affected by the loss of AhpC is an unusual phenomenon, unrelated to iron storage, which is different from the modulation of catalase activity by BfrA in P. aeruginosa. As shown in Fig. 1, the resolution of the KatA protein band and its density relative to the total protein pattern allow it to be monitored unambiguously. A comparison of the protein profiles from crude extracts of the wild type with various mutant strains (Fig. 1) indicated that the knock-out of AhpC did not change significantly the level of catalase protein expression. Hence, a regulatory role for AhpC in catalase expression was ruled out. Therefore, we hypothesized that the catalase in ahpC mutant cells might have undergone certain structural changes leading to its partial inactivation. Purified H. pylori AhpC was shown to be able to reduce H2O2in vitro at a rate similar to its t-BOOH reducing activity (22Baker L.M. Raudonikiene A. Hoffman P.S. Poole L.B. J. Bacteriol. 2001; 183: 1961-1973Crossref PubMed Scopus (162) Google Scholar). However, the H2O2 decomposing activity in the katA mutant (AhpC+) cells was undetectable (our results are shown in Fig. 2A) (see Refs. 23Odenbreit S. Wieland B. Haas R. J. Bacteriol. 1996; 178: 6960-6967Crossref PubMed Google Scholar and 24Harris A.G. Hinds F.E. Beckhouse A.G. Kolesniow T. Hazell S.L. Microbiology. 2002; 148: 3813-3825Crossref PubMed Scopus (69) Google Scholar). Because AhpC requires thioredoxin and thioredoxin reductase for its activity, failure to detect the H2O2 decomposing activity of AhpC could be the result of a limited amount of available thioredoxin-thioredoxin reductase proteins. Therefore, we tested whether the wild type cell extract has the activity to reduce organic peroxides. Using t-BOOH as a substrate and without adding reductant, the wild type cell extract showed reductase activity, with a specific activity of 15.6 ± 2.3 nmol (t-BOOH reduced)/min/mg of total protein in the cell extract. If AhpC had a similar rate of H2O2 reducing activity, it would be far below the detection level of the assay system for catalase. Thus, the decrease of catalase activity observed in ahpC mutant cells could not be attributed to the loss of the H2O2 decomposing activity of AhpC but to the perturbation of catalase itself. A Perturbed Heme Environment Associated with Catalase in ahpC Mutant Cells—Catalase contains a heme-active site, which can be detected by EPR spectroscopy in ferric oxidation states (36Nicholls P. Fita I. Loewen P.C. Adv. Inorg. Chem. 2001; 51: 51-106Crossref Google Scholar). Purified H. pylori catalase exhibits a characteristic, nearly axial high spin ferric EPR signal in which effective g values = 6.36, 5.35, and 1.98 (37Loewen P.C. Carpena X. Rovira C. Ivancich A. Perez-Luque R. Haas R. Odenbreit S. Nicholls P. Fita I. Biochemistry. 2004; 43: 3089-3103Crossref PubMed Scopus (67) Google Scholar). Considering the high abundance of catalase protein in H. pylori and the high transition probability of the low field components of axial high spin ferric resonances, we applied EPR analysis to whole cells to mon