Original Contribution

STREPTOCOCCUS MUTANSH2O2-FORMING NADH OXIDASE IS AN ALKYLHYDROPEROXIDE REDUCTASE PROTEIN

LESLIE B. POOLE,* M ASAKO HIGUCHI,† MAMORU SHIMADA ,‡ MARCO LI CALZI ,* and YOSHIYUKI KAMIO†

*Department of Biochemistry, Wake Forest University School of Medicine,Winston-Salem, NC, USA;†Department of Molecularand Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan; and‡Research Center, Nippon Paint

Co., Ltd., Osaka, Japan

(Received18 August1999;Accepted18 October1999)

Abstract—Nox-1 from Streptococcus mutans, the bacteria which cause dental caries, was previously identified as anH2O2-forming reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nox-1 is homologous with the flavoproteincomponent, AhpF, ofSalmonella typhimuriumalkyl hydroperoxide reductase. A partial open reading frame upstreamof nox1, homologous with the other (peroxidase) component,ahpC, from theS. typhimuriumsystem, was also identified.We report here the complete sequence ofS. mutans ahpC. Analyses of purified AhpC together with Nox-1 have verifiedthat these proteins act as a cysteine-based peroxidase system inS. mutans, catalyzing the NADH-dependent reductionof organic hydroperoxides or H2O2 to their respective alcohols and/or H2O. These proteins also catalyze the four-electron reduction of O2 to H2O, clarifying the role of Nox-1 as a protective protein against oxygen toxicity. Majordifferences between Nox-1 and AhpF include: (i) the absolute specificity of Nox-1 for NADH; (ii) lower amounts offlavin semiquinone and a more prominent FADH2 to NAD1 charge transfer absorbance band stabilized by Nox-1; and(iii) even higher redox potentials of disulfide centers relative to flavin for Nox-1. Although Nox-1 and AhpC fromS.mutanswere shown to play a protective role against oxidative stress in vitro and in vivo inEscherichia coli, the lackof a significant effect on deletion of these genes fromS. mutanssuggests the presence of additional antioxidant proteinsin these bacteria. © 2000 Elsevier Science Inc.

Keywords—Alkyl hydroperoxide reductase, NADH oxidase, Disulfide reductase, Flavoprotein, Redox centers, Perox-idase, Peroxiredoxin, Free radicals

INTRODUCTION

Streptococcus mutans, like other types of lactic acidbacteria, lack catalase and a functional electron-transportchain and are thus dependent on fermentative metabo-lism and alternative pathways for aerobic growth [1].Various strains ofS. mutans, a causative agent of dentalcaries, vary in their ability to grow aerobically; aerotol-erance of these strains has been shown to correlate withtheir ability to induce superoxide dismutase and reducednicotinamide adenine dinucleotide (NADH) oxidase[2,3]. On further investigation, two distinct NADH oxi-dase enzymes were identified in an O2-tolerantS. mutansstrain, and these enzymes were shown to catalyze either

the two-electron reduction of O2 to H2O2 (Nox-1) or thefour-electron reduction of O2 to H2O (Nox-2) [3,4].Sequence analyses have confirmed that these two fla-voproteins are only distantly related. Nox-1 is homolo-gous with a group of bacterial enzymes, several of whichhave been shown to possess H2O2-forming NADH oxi-dase activity and dehydrogenase activity, which supportshydroperoxide reduction by a second protein, AhpC[5–7]. Nox-2 is homologous with several H2O-formingNADH oxidases exemplified by the enzyme fromEn-terococcus faecalis[8,9].

Sequence-based searches for proteins homologouswith AhpF and AhpC, the two components of alkylhydroperoxide reductase fromSalmonella typhimurium,have indicated a very wide distribution of the peroxide-reducing protein, AhpC, in organisms ranging from ar-chaebacteria to plants and mammals, whereas close ho-mologues of the flavoenzyme, AhpF, have been found

Address correspondence to: Leslie B. Poole, Department of Bio-chemistry, Wake Forest University School of Medicine, Medical Cen-ter Boulevard, Winston-Salem, NC 27157, USA; Tel: (336) 716-6711;Fax: (336) 716-7671; E-mail: lbpoole@wfubmc.edu.

Free Radical Biology & Medicine, Vol. 28, No. 1, pp. 108–120, 2000Copyright © 2000 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/00/$–see front matter

PII S0891-5849(99)00218-X

108

only in eubacteria [6,7]. AhpF homologues are distantlyrelated to thioredoxin reductase (# 36% amino acididentity), and retain flavin-binding, pyridine nucleotide-binding and redox-active cystine motifs, but also includea region of approximately 200 amino acids at their N-terminus, which is not present in thioredoxin reductase[7,10]. Structure–function studies ofS. typhimuriumAhpF have demonstrated that this “appended” N-termi-nal region, as well as an additional redox-active cystinecenter contained within it, are required for reduction ofAhpC and thus peroxides [7,11,12].

In each bacterial sequence where a full length AhpFhomologue has been identified, a full-length or partialopen reading frame encoding an AhpC-like protein hasbeen identified upstream of the structural gene for theflavoprotein, strongly suggesting that these flavoproteinsare all linked with peroxidase systems [6,7,13]. Peroxi-dase activities for systems other than that fromS. typhi-murium have been confirmed in several cases [13–17].Mechanistic studies focusing on the oxidase activity ofthe relatedAmphibacillus xylanusNADH oxidase fla-voprotein have been reported [18], although only prelim-inary information regarding the activity of this flavopro-tein with its own AhpC is available [15]. We havetherefore undertaken a detailed analysis of the Nox-1/AhpC system ofS. mutansto establish the similaritiesand differences between these systems for gram-negativeand gram-positive organisms.

We report here the full sequence of theahpC geneupstream ofnox1 in S. mutans, as well as the expressionand characterization of both proteins now known to forman organic hydroperoxidase system. Our investigationshave includedS. typhimuriumAhpF and AhpC proteinsin heterologous catalytic assays, and have permitted de-tailed comparisons of the functional properties of theseproteins from gram-positive and gram-negative bacteria.

MATERIALS AND METHODS

Molecular biological techniques

Subcloning ofnox1 to create pNx1H2 for overexpres-sion of Nox-1 protein was accomplished by ligating the1.6 kbHindIII-HindIII fragment from pNox1-H [19] intothe corresponding site of pPROK-1 (Clontech, Palo Alto,CA, USA) following standard protocols [20] with en-zymes from New England Biolabs (Beverly, MA, USA).S. mutans ahpCwas found to be entirely encoded withinpMS1, a pUC119-derived plasmid containing a 1.9 kbEcoR1-EcoR1 fragment from the originall clone con-taining nox1, lHS-1 [19]. This fragment was amplifiedby the polymerase chain reaction (PCR) using primersCCGAATTCAGGAGGAAGTATAGATGTCTTTAGT-CGG and CCCTGCAGGCCATAGTTTCTCCTTAAA-

TTTTACCGACAAGTACC (underlined regions indi-cate addedEcoRI and PstI sites, respectively, anengineered ribosome binding site is shown in italics, andthe ATG start codon is shown inbold), vent polymerasefrom New England Biolabs, and standard PCR condi-tions suggested by the manufacturer. Restriction di-gestion withEcoRI and PstI and ligation of the di-gested fragment into the corresponding sites ofpPROK-1 created the AhpC overexpression vectorpSMAC-1.

Protein purifications and mass spectrometry

General methods for maintaining and culturingEsch-erichia coli strains and carrying out the purification ofrecombinant AhpF and AhpC proteins fromS. typhi-muriumhave been described previously [21]. For theS.mutansproteins, cultures ofE. coli strain JM105 harbor-ing the appropriate expression plasmid (pNx1H2 orpSMAC-1) were grown in 10 l of ampicillin-containingLuria broth supplemented with 0.2% glucose in a BioFlo2000 fermentor (New Brunswick Scientific Co., Inc.,Edison, NJ, USA) after inoculation with 2% of an over-night culture. Isopropyl b-D-thiogalactopyranoside(IPTG) was added at an A600 of 1.0–1.5, and the growthwas continued overnight. Harvested cell pellets werestored at220°C until needed. For purification of AhpC,streptomycin sulfate-treated cell extracts from mechani-cal disruption were brought to 50% ammonium sulfate inthe standard buffer (25 mM potassium phosphate bufferat pH 7.0, containing 1 mM ethylenediaminetetraacetate[EDTA]), pelleted protein was resuspended in the samebuffer containing 30% ammonium sulfate, and the pro-tein was loaded, washed, and eluted from a PhenylSepharose 6 Fast Flow Column as previously described[21], except that the initial concentration of 30% ammo-nium sulfate was used instead of 20%;S. mutansAhpCelutes at about 23% ammonium sulfate. After overnightdialysis versus two changes of standard phosphatebuffer, pooled protein was loaded onto a DE52 columnpre-equilibrated in 0.15 M KCl-containing buffer,washed with the same buffer, and eluted with a gradientof 0.15–0.35 M KCl; under these conditions AhpC elutesat about 0.2 M KCl. In some cases, a Q Sepharose FPLCcolumn was used instead of DE52 with buffers contain-ing the same salt concentrations. Fractions containingpure AhpC were pooled, dialyzed and stored at220°Cuntil further use.

Nox-1 was purified in a similar way, except that thecrude extract was treated only with 20% ammoniumsulfate and loaded directly on a Phenyl Sepharose col-umn with elution from 20 to 0% ammonium sulfate;Nox-1 elutes at about 14% ammonium sulfate (S. typhi-murium AhpF does not elute from this column even

109S. mutansperoxide reductase

when washed extensively with the standard phosphatebuffer lacking ammonium sulfate). After overnight dial-ysis, the Nox-1 pool was loaded and eluted from a DE52or Q Sepharose column with a 0.1–0.3 M KCl gradient.If necessary, the protein pool was further purified with aBio-Gel A-0.5 m agarose column as previously described[22]. Unlike AhpF, Nox-1 does not bind to Affi-Gel Blue[21,22]. The major contaminant of Nox-1, when present,is apparentlyE. coli AhpC; expression of Nox-1 in theTA4315 strain of E. coli lacking ahpCF avoids thisproblem [10].

Pure protein samples were washed free of phos-phate buffer and EDTA and analyzed by electrosprayionization mass spectrometry (ESI MS) as previouslydescribed [23].

Assays and titrations

Spectrophotometric assays were performed essen-tially as described previously [21], with some modifica-tions. Buffers used in transhydrogenase, 5,59-dithiobis(2-nitrobenzoic acid) (DTNB) reductase and peroxidaseassays contained 100 mM ammonium sulfate, and allassays were carried out at 25°C. A Gilford 260 recordingspectrophotometer (Gilford Instrument Laboratories,Inc., Oberlin, OH, USA) was used for oxidase and tran-shydrogenase assays, and an Applied Photophysics(Leatherhead Surrey, UK) DX.17MV stopped-flow spec-trophotometer was used for DTNB reductase and perox-idase assays. Crude extracts were assayed anaerobicallyfor flavoprotein- or AhpC-dependent peroxidase activityas described previously [19]. Some oxidase assays em-ployed a Model 5/6H oxygraph (Gilson Medical Elec-tronics, Inc., Middletown, WI, USA) with a Clark oxy-gen electrode (Yellow Springs Instrument Co., YellowSprings, OH, USA) and a 2 ml waterjacketed samplechamber. Anaerobic titrations, thiol quantitation usingDTNB, microbiuret assays for determination of proteinconcentrations, and determination of extinction coeffi-cients were all performed as previously described [7, 21]using a thermostatted Spectronic 3000 diode array spec-trophotometer (Milton Roy Co., Rochester, NY, USA)with 0.35 nm resolution.

Sequence analyses

DNA sequence analyses of the coding regions of allplasmids were carried out by the DNA sequence analysiscore laboratory of the Comprehensive Cancer Center ofWake Forest University using a Prism 377 automatedDNA sequencer (Applied Biosystems, Inc., Foster City,CA, USA). Analysis of the DNA sequence upstream ofahpCwas carried out to locate putative ribosome binding

and promoter sequences [24,25]. TERMINATORsearches, pairwise comparisons (BESTFIT) and multiplesequence alignments (PILEUP) were performed usingthe Genetics Computer Group suite of programs (Uni-versity of Wisconsin) and the default parameters. Mul-tiple sequence alignments included sequences from thefollowing organisms (GenBank accession numbers orreference in parentheses):S. mutans(this work),S. pyo-genes(Roe, Clifton, McShan and Ferretti, StreptococcalGenome Sequencing Project at the University of Okla-homa), Bacillus subtilis (D78193), B. alcalophilus(JX0166),Amphibacillus xylanus(AB018435),Staphy-lococcus aureus(X85029), Salmonella typhimurium(J05478), E. coli (D90701), Pseudomonas putida(AB010689), andXanthomonas campestris(U94336).Pairwise comparisons also included the following:En-terococcus faecalis(AF016233),Clostridium pasteuria-num(M60116),Legionella pneumophila(L46863),Hel-icobacter pylori (AE000654), Campylobacter jejuni(AF044271),Rickettsia prowazekii(AJ235271),Myco-bacterium tuberculosis (U16243), Corynebacteriumdiphtheriae (U18620), Aquifex aeolicus(AE000692),Synechocystisspecies (D64000),Chlamydia pneumoniae(AE001659), andC. trachomatis (AE001331). TheDDBJ accession number forS. mutanssequences re-ported in this manuscript is AB010712.

RESULTS

Subcloning, expression and purification ofS. mutansNox-1 and AhpC

The expression of Nox-1 from its own promoter usingthe low copy plasmid pMW118 was previously reportedto result in levels of this protein 2-fold over those of thenative protein inS. mutans[5]. A new vector, pNx1H2,was generated for expression of Nox-1 under the controlof the tac promoter, and crude extracts of IPTG-inducedE. coli strain JM105 harboring this construct containedNox-1 at about 30% of the total protein. Sequence anal-yses of pNx1H2, as well as plasmids derived from theoriginal l clone containingnox1 genomic sequence,lHS-1 [5], confirmed the absence of inadvertent muta-tions in the PCR-generated expression plasmid and,based on resequencing of the genomic clone, led toseveral corrections of the originally reported nucleotideand deduced amino acid sequences (Fig. 1). The enzymewas obtained in its purified form using a protocol relatedto those previously reported forS. mutansNox-1 andS.typhimuriumAhpF (Fig. 2) [5,21]. The molecular massof the purified protein as determined by ESI MS was55,020 per subunit, comparing favorably with the ex-pected mass of 55,012 for the apoprotein lacking theinitiating methionine. Previous N-terminal sequence re-

110 L. B. POOLE et al.

sults of Nox-1 isolated fromS. mutansalso indicated theremoval of the initiating methionine [5]. In contrast, theN-terminal methionine ofS. typhimuriumAhpF is notremoved [22].

To obtain the complete sequence of theahpC geneupstream ofnox1, a plasmid containing the 1902 bpEcoRI-EcoRI fragment from the originall clone wassequenced and found to contain the entire coding regionfor AhpC as well as 345 bp upstream of the structuralgene (Fig. 1). As for Nox-1, an expression plasmid,pSMAC1, was generated for expression of AhpC underthe control of thetac promoter, giving expression levelsof about 30% for AhpC in crude extracts of IPTG-induced JM105 harboring this construct. Purification ofS. mutansAhpC was accomplished after a protocol verysimilar to that previously established forS. typhimuriumAhpC (Fig. 2) [21]. This procedure gave a yield of morethan 600 mg of pure protein from 10 l of bacterialculture. The mass of AhpC by ESI MS was 40,685,

nearly identical with the predicted mass of 40,690 for thedimeric protein lacking the initiating methionine residue.This result, as well as the migration of the oxidizedprotein as a covalent dimer on a nonreducing, denaturingSDS-polyacrylamide gel (not shown), confirms the pres-ence of intersubunit disulfide bonds in AhpC. Althoughthe primary structure ofS. mutansAhpC has not beenanalyzed by N-terminal sequencing, removal of the ini-tiating methionine has also been observed forS. typhi-muriumAhpC [21,22].

Spectral properties and thiol contents ofS. mutansalkyl hydroperoxide reductase proteins

Nox-1 possesses one tightly-bound flavin adeninedinucleotide (FAD) per subunit with spectral propertiestypical of a flavoprotein reductase. The extinction coef-ficient of this bound FAD at 448 nm, like that of AhpF,is higher than that of free flavin, at 13,0506 250 M21

Fig. 1. Nucleotide and deduced amino acid sequences ofS. mutans ahpCandnox1 loci. Putative promoter (235 and210) and ribosomebinding sites (rbs) are underlined, two inverted regions forming possible terminators are indicated by arrows, putative active sitehalf-cystines are boxed, and stop codons are indicated by asterisks. Initiator methionines indicated in parentheses have been shown bymass spectrometric analyses to be removed from the mature proteins.

111S. mutansperoxide reductase

cm21 (n 5 6). The other flavin peak (lmax 5 382 nm)has an even higher extinction coefficient, at 13,800 M21

cm21 (Fig. 3), whereas the 380 nm peak of AhpF islower than that at 450 nm (e 5 12,600 M21 cm21) [7].

Four cysteine residues in the deduced amino acidsequence of Nox-1, at positions 127, 130, 336, and 339,align perfectly with the half-cystines associated with thetwo redox-active disulfide bonds in AhpF (see sequencecomparisons, below). One additional cysteine, at position480, is present in 7 of the 10 bacterial homologuesidentified to date and aligns with Cys489 of AhpF, aresidue which was previously shown by site-directedmutagenesis to play no structural or catalytic role in thatprotein [7]. Oxidized Nox-1 contains one inaccessiblethiol per subunit as detected by addition of DTNB in thepresence or absence of denaturant (Table 1). The thioltiter increases by four per subunit if the protein is firstreduced with excess NADH consistent with the partici-pation of the other four cysteines in two redox-activedisulfide centers, as is the case for AhpF.

S. mutansAhpC, like its counterpart fromS. typhi-murium, possesses no chromophoric cofactor and is notinactivated on dialysis in the presence of Chelex-100 toremove protein-bound metals. A 1 mg/ml solution ofpureS. mutansAhpC gave an absorbance of 1.16 at 280nm, nearly identical with the value of 1.18 determinedfor S. typhimuriumAhpC [21]. Two cysteine residues, atpositions 46 and 164, also align perfectly with the twocatalytic cysteines at positions 46 and 165 inS. typhi-muriumAhpC. One additional cysteine, present only inS. mutansAhpC, is located at position 36 and apparentlyexists as an inaccessible free thiol in the oxidized andreduced proteins (Table 1). Consistent with the expectedredox-active nature of the intersubunit disulfide bondbetween Cys46 and Cys1649 of S. mutansAhpC, twonew highly accessible thiols per monomer are generatedon reduction of the protein in the presence of catalyticamounts of Nox-1 and excess NADH.

Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ofrecombinantS. mutansNox-1 and AhpC proteins expressed inE. coli,at various stages of purification. The 0.75 mm-thick 12% acrylamidegel was run at 35 mA with samples pretreated by boiling for 3 min insample buffer containing 2% sodium dodecyl sulfate and 5% 2-mer-captoethanol. Lane 1 contains low-range protein standards from Bio-Rad with indicated Mr values. Lanes 2 and 5 contain 25mg samples ofcrudeE. coli extracts from IPTG-induced JM105 harboring pNx1H2 orpSMAC-1, respectively. Approximately 8mg each of Nox-1 or AhpC-containing samples were loaded in lanes 3 and 4, or in lanes 6 and 7,respectively, following phenyl sepharose (lanes 3 and 6) and DEAE-cellulose chromatography (lanes 4 and 7).

Fig. 3. Comparison ofS. mutansNox-1 andS. typhimuriumAhpFspectra with and without excess NADH. Spectra shown are Nox-1(solid lines) and AhpF (dashed lines) in the absence (high A448) orpresence (low A448, high A800) of 20 equiv of NADH/FAD addedunder anaerobic conditions.

Table 1. Thiol Quantitation ofS. mutansNox-1 and AhpC ProteinsBefore and After Reduction With NADHa

Oxidized protein Reduced proteinb

No denaturantc1 4 MGuHCl No denaturantc

1 4 MGuHCl

Nox-1 0.026 0.01 1.016 0.04 N.D.d 4.996 0.35AhpC 0.076 0.01 0.966 0.01 1.906 0.10 2.906 0.01

a Each experiment was performed at least three times; values shownare mean6 standard error and are reported per subunit for each protein.

b Each protein was reduced anaerobically with a 5-fold molar excessof NADH; a catalytic amount of Nox-1 (1/100 equivalent) was alsoincluded in the experiments with AhpC.

c Values were obtained after 60-min incubation with DTNB; becauseof partially reduced state of AhpC as isolated, the protein was prein-cubated with H2O2 and reconcentrated with Centricon CM-30 ultrafil-tration units (Amicon, Inc., Beverly, MA, USA) before assay.

d Not determined; addition of DTNB in the presence of excessNADH but no denaturant results in turnover.

112 L. B. POOLE et al.

Nox-1 catalytic activities in the presence and absenceof AhpC

Nox-1 was previously identified as an H2O2-produc-ing NADH oxidase, an activity also known to be exhib-ited by related flavoproteins fromA. xylanusand S.typhimurium[4,14,26]. In the presence of excessS. ty-phimuriumAhpC, S. typhimuriumAhpF or A. xylanusNADH oxidase also supported the full four-electron re-duction of oxygen to water [14]. A direct comparison ofthe oxidase activities ofS. typhimuriumAhpF andS.mutansNox-1 determined spectroscopically in the pres-ence of 260mM oxygen indicated approximately 5-foldhigher activity for theS. mutansflavoprotein relative tothat of S. typhimurium(Table 2). In both cases theoxidase activity is increased on addition of free FAD.This additional activity results from the ability of theseflavoproteins to catalytically reduce free FAD; oxygenthen reacts with the free FADH2 nonenzymatically (datanot shown).

Oxidase assays were then performed using an oxygenelectrode and limiting NADH to demonstrate the con-version of Nox-1 from an H2O2-producing oxidase sys-tem to an H2O-producing system on addition of theAhpC component, as indicated by the presence or ab-sence of O2 regeneration at 1/2 eq on addition of catalaseat the end of the reaction (Fig. 4). Because our assayconditions included NADH in limiting concentrationsrelative to O2, we were also able to demonstrate that thisconversion was accompanied by the expected change instoichiometry of NADH:O2 consumed from 1:1 to 2:1 onaddition of AhpC (Fig. 4, Scheme 1). On varying theAhpC concentration, intermediate ratios of NADH:O2

are obtained because of the competition between the two

activities (oxidase activity of Nox-1 alone or peroxidaseactivity of Nox-1 plus AhpC) for the limited supply ofNADH. Any change in conditions favoring one activityover the other shifts the amount of AhpC needed toachieve an intermediate ratio (e.g., 1.5 NADH:O2). Thisassay was used to show an activation of peroxidaseactivity but not oxidase activity on addition of ammo-nium sulfate, as was previously demonstrated for AhpF

Fig. 4. Oxygen consumption during oxidase assays of Nox-1 in thepresence or absence of AhpC using limiting NADH. Panel A, thereaction mixture contained Nox-1 alone (1 nmol) in phosphate buffer(pH 7) containing 100 mM ammonium sulfate and was initiated by theaddition of 200mM NADH; catalase (80mg) was added after theconsumption of oxygen ceased. Panel B, the reaction was carried out asin Panel A, but included 1 nmol AhpC.

Table 2. Comparison of Catalytic Activities ofS. mutansNox-1 andS. typhimuriunAhpFa

AhpF Nox-1

Oxidaseb 1.866 0.06 8.696 0.45Transhydrogenaseb 56.56 1.3 61.86 3.5DTNB reductasec

Vmax 26.96 1.5 31.66 2.0Km for NADH 1.146 0.20 1.536 0.33

a Values given as mean6 standard error; Vmax expressed as micro-moles NADH consumed s21 micromole21 bound FAD, Km asmM.

b Oxidase and transhydrogenase assays were performed least eighttimes and included the following substrates: NADH at 150mM, oxygenat 260mM, and oxidized 3-acetyl pyridine adenine dinucleotide at 150mM (transhydrogenase). Oxidase assays included 30–60 pmol AhpF or5–15 pmol Nox-1; transhydrogenase assays included 2.2–7 pmol ofeither flavoprotein.

c DTNB turnover assays with NADH were performed using astopped-flow spectrophotometer with 20 nM of either flavoprotein, 500mM DTNB, and NADH varied from 0.2–20mM, in three independentdeterminations. Curve fitting to the Michaelis Menten equation wascarried out using the program ENZFITTER.

Scheme 1.

113S. mutansperoxide reductase

[21]. In the presence of 500 nM or less of AhpF, additionof 100 mM ammonium sulfate to the assay buffer dropsthe amount ofS. typhimuriumAhpC needed to achieve aratio of 1.5 NADH:O2 from 125 to 75 nM; with 200 nMor less of Nox-1, theS. mutansAhpC concentration for1.5 NADH:O2 drops from about 1mM to 150 nM in thepresence of ammonium sulfate. Thus, the AhpC concen-tration needed to achieve a balance in NADH utilizationfor oxidase and peroxidase activities in the presence ofammonium sulfate is about twice as high for theS.mutansalkyl hydroperoxide reductase system relative tothat of S. typhimurium, in part because of the higheroxidase activity of Nox-1; the activating effect of am-monium sulfate on the peroxidase activity is also sub-stantially greater for theS. mutanssystem.

Peroxidase activities assessed by anaerobic assayswith AhpC in excess over flavoprotein indicated verysimilar kinetic parameters for theS. mutansNox-1/AhpCsystem compared with AhpF/AhpC fromS. typhi-murium, although the Vmax for the reaction was a littlelower in the former case (Table 3). As shown previouslyfor the S. typhimuriumperoxidase system [14], theseassays produced identical results when carried out aero-bically in spite of the higher oxidase activity of Nox-1.Interestingly, such assays performed on the stopped flowspectrophotometer indicate that the initial rate in theabsence of peroxide, attributed to the reduction of AhpC,mimics the rate of reaction where peroxide is present,indicating that AhpC turnover with peroxide is at least asfast as the transfer of electrons to AhpC under the con-ditions used (Fig. 5). The same assays performed using aheterologous mixture of the two proteins from the twodifferent bacteria gave very similar values to those foreach system (Table 3), indicating a high degree of sim-ilarity between the way each of the protein componentsinteract for catalysis.

Two other activities catalyzed by AhpF [21], NADH:AcPyAD1 oxidoreductase (transhydrogenase) activity,during which a hydride ion is transferred from one pyri-dine nucleotide to another, and NADH:DTNB oxi-doreductase (DTNB reductase) activity, during which a

disulfide is reduced to generate a chromaphoric thiol-containing product, are also catalyzed with high turnovernumbers by Nox-1 (Table 2). The Km for NADH using500mM DTNB as the electron acceptor is also very lowand similar for both AhpF and Nox-1, around 1–2mM.Unlike AhpF, which is capable of using NADPH as asubstrate (albeit with a Km more than two orders ofmagnitude higher than that for NADH) [21], no DTNBreductase activity was observed for Nox-1 usingNADPH.

Reductive titrations ofS. mutansalkyl hydroperoxidereductase proteins

To firmly establish the stoichiometry and identity ofredox centers within eachS. mutansalkyl hydroperoxidereductase protein, anaerobic reductive titrations werecarried out similar to those previously reported for theS.typhimuriumAhpF and AhpC proteins [7]. Like AhpF,Nox-1 is fully reduced by approximately three eq ofdithionite per FAD, indicating the presence of two otherredox centers in addition to the FAD in each subunit(Fig. 6). Some degree of stabilization of the one-electronreduced blue, neutral semiquinone form of the flavin isalso observed during reductive titrations, as evidenced bythe peak observed around 580 nm for the partially re-duced protein [27]. Taking the reported extinction coef-ficient of 4790 M21 cm21 at 580 nm for this form of theflavin in the S. typhimuriumprotein as an approximatevalue for this species in Nox-1 [7], the maximum extentof semiquinone formation during dithionite titrations ofNox-1 is approximately 30% (relative to;91% for

Fig. 5. NADH oxidation byS. mutansNox-1 and AhpC in the presenceor absence of cumene hydroperoxide. Nox-1 (500 nM) and AhpC (28mM) in peroxidase assay buffer (pH 7) were mixed with assay buffercontaining 150mM NADH with (circles) or without (squares) 1 mMcumene hydroperoxide (final concentrations). The same data (lines) isshown over different time courses in the main plot and inset, withsquares or circles at every 20th data point.

Table 3. Peroxidase Activity ofS. mutansandS. typhimuriumAlkylHydroperoxide Reductase Proteinsa

S. mutansAhpC S. typhimuriumAhpC

S. mutansNox-1 Vmax 1666 11 1806 8Km 18.46 2.7 21.26 1.9

S. typhimuriumAhpF Vmax 2136 15 2376 17Km 26.66 3.7 19.06 2.8

a Values given as mean6 standard error; Vmax expressed as micro-moles NADH consumed s21 z micromole21 bound FAD, Km asmM;assays performed anaerobically with [AhpC] varied from 3 to 50mMin the presence of 0.5mM flavoprotein, 200mM NADH, and 1 mMcumene hydroperoxide.

114 L. B. POOLE et al.

AhpF). Indeed, combination of spectra accounting for35, 30, and 35%, respectively, of FAD, FADH•, andFADH2 as determined for AhpF give a spectrum reason-ably similar to that of Nox-1 after addition of 2.42 eq ofdithionite except for a small additional absorbancearound 520 nm (Fig. 6, dotted spectrum). Other flavopro-tein dehydrogenases exhibiting charge-transfer interac-tions between a nascent thiolate anion and oxidized fla-vin also show a shoulder of absorbance around 530–540nm, but this species has not been clearly identified inNox-1 or AhpF.

Titration of Nox-1 with NADH also leads to reductionof the two nonflavin redox centers and partial reductionof the flavin, as is the case with AhpF, but again theextent of flavin semiquinone formation is less than thatduring NADH titration of AhpF (Fig. 7) [7]. Consump-tion of NADH during the titration as monitored at 354nm (an approximate isosbestic point for flavin absor-bance changes up to 2.5 eq of added dithionite; Fig. 6) isapproximately 3 eq per FAD. An additional long wave-length absorbance band beyond 700 nm is generatedduring NADH, but not dithionite, titrations and is attrib-utable to the formation of a FADH2 3 NAD1 chargetransfer species (Figs. 3 and 7). On addition of 20 eq ofNADH, the apparent extinction coefficient for this spe-cies of Nox-1 at 763 nm is about 1600 M21 cm21

(relative to 650 M21 cm21 for AhpF; Fig. 3) [7].NADH titration of S. mutansAhpC in the presence of

a catalytic amount of Nox-1 leads to the consumption ofapproximately 1 eq of NADH per subunit of AhpC andthe generation of two new free thiol groups, as previ-ously demonstrated forS. typhimuriumAhpC (Table 1)[7]. Consistent with the hypothesized roles forS. mutans

Nox-1 and AhpC in catalysis of peroxide reduction(Scheme 1), addition of 3 eq of AhpC to NADH-reducedNox-1 results in nearly full regeneration of the oxidizedspectrum of this protein, whereas 1 eq of ethyl hydroper-oxide per subunit is sufficient for reoxidation of two ofthe free thiols ofS. mutansAhpC back to a disulfidebond.

Comparisons ofS. mutansalkyl hydroperoxidereductase protein sequences with those of otherorganisms

Alignments of bacterial proteins related to Nox-1and AhpF indicate a high degree of sequence conser-vation surrounding motifs for NADH and FAD bind-ing and for the redox-active half-cystine residues ofAhpF (Fig. 8) [7]. AhpC homologues corresponding tothose bacteria that possess AhpF homologues as de-scribed above have been aligned as shown in Fig. 9.These bacterial AhpC proteins exhibit a very highdegree of sequence conservation, particularly sur-

Fig. 6. Anaerobic dithionite titration of Nox-1. Solid lines representspectra obtained after all absorbance changes were complete followingadditions of 0, 0.77, 1.30, 1.86, and 2.42 eq of dithionite/FAD, in orderof decreasing A448and increasing A580. The dashed line is the spectrumobtained after addition of 3.0 eq of dithionite/FAD. The dotted lineshows a calculated spectrum representative of 35% oxidized, 30%semiquinone, and 35% fully reduced forms ofS. typhimuriumAhpF.The inset shows the absorbance changes at 448 (closed squares) and580 nm (open circles) vs. equiv of dithionite/FAD added.

Fig. 7. Anaerobic NADH titration of Nox-1. Solid lines representspectra obtained after addition of 0, 0.87, 1.53, 2.19, 2.84, and 3.50 eqof NADH/FAD, in order of decreasing A448 and increasing A580.Dashed lines are the spectra obtained after addition of 4.15 and 4.81 eqof NADH/FAD, in order of increasing A340 and A800. The inset showsthe absorbance changes at 354 (open squares), 448 (closed squares),580 (open circles), and 750 nm (closed circles) vs. equiv of NADH/FAD added.

115S. mutansperoxide reductase

rounding the catalytic half-cystines (at positions 46and 165/164 inS. typhimuriumand S. mutanspro-teins). Out of the 6 gram-positive and 4 gram-negative

sequences compared, 68 of the 186 –189 positions areabsolutely conserved. Biochemical data from our stud-ies of S. typhimuriumAhpC have established the re-

Fig. 8. Multiple sequence alignments of Nox-1/AhpF homologues. The consensus sequence shows fully identical amino acids in uppercase and those with one or two exceptions in lower case. The active-site half-cystine residues as identified inS. typhimuriumAhpF areshown in bold. Motifs (GxGxxG) for flavin and NAD(P)H binding are indicated by single and double underlines, respectively.

116 L. B. POOLE et al.

quirement for a single active site cysteine, Cys46, tocatalyze peroxide reduction through formation of acysteine sulfenic acid on the enzyme; the second cys-teine, Cys165, is conserved among most homologuesand is important in preventing the inactivation ofAhpC (resulting from oxidation of the Cys46 sulfenicacid by peroxides or oxygen) through formation of ahighly unusual redox-active disulfide bond betweentwo different subunits at the active site [23,28].

Multiple pairwise comparisons of all known bacte-rial AhpC homologues including those without AhpFstructural genes downstream ofahpC (Table 4) showgreater divergence of the (apparently) AhpF-indepen-dent AhpC homologues from the ones shown in Fig. 8.The notable exception to this is the homologue fromAquifex aeolicus,which shows an unusual degree ofsimilarity to the AhpF-linked AhpC homologues. TwoahpC genes, those fromEnterococcus faecalisandClostridium pasteurianum, are in close proximity to atrxB homologue lacking the region corresponding tothe N-terminal segment of AhpF, and those deducedAhpC sequences are also more divergent relative tothe AhpF-linked AhpCs (Table 4). Both redox-activehalf-cystine residues identified inS. typhimuriumandS. mutansAhpC are conserved among the bacterialAhpCs included in Table 4; two of these bacteria,Synechocystissp. andAquifex aeolicus, also encode asecond AhpC homologue within their genome withonly the Cys46 conserved.

DISCUSSION

Nox-1 fromS. mutans, a protein originally isolated asan NADH oxidase, has now been demonstrated to func-tion as the flavoprotein dehydrogenase component of analkyl hydroperoxide reductase system; the peroxidasecomponent, AhpC, is encoded directly upstream ofnox1in theS. mutansgenome. Although Nox-1 and an NADHoxidase fromAmphibacillus xylanus[26] were originallyidentified based on their oxidase activity (which is about5-fold higher than that ofS. typhimuriumAhpF), H2O2

formation by these flavoproteins clearly distinguishesthem from the H2O-forming oxidases (e.g., Nox-2 fromS. mutansand NADH oxidase fromEnterococcus faeca-lis), which play an important metabolic role in recyclingNADH for glycolysis in these heme-deficient (and there-fore electron-transport chain deficient) organisms [4,9,19]. Our studies have clearly shown that the turnover ofNox-1 and AhpF with oxygen at 25°C is significantlyslower than turnover with peroxides in the presence ofexcess AhpC by 20–100-fold (Tables 2 and 3); additionof excess AhpC in the oxygen electrode experiments wasalso shown to result in formation of H2O rather thanH2O2 by the oxidase activity of Nox-1 and AhpF.Whereas previous investigations that detected H2O2 for-mation by Nox-1 seemed to be in conflict with a positiverole for this enzyme in aerobic metabolism [4,5], ourdemonstration of the function of Nox-1 as part of analkyl hydroperoxide reductase system in combinationwith AhpC clarifies its role as an antioxidant.

Fig. 9. Multiple sequence alignments of AhpC homologues. Aligned protein sequences of AhpC homologues from the same organismsas in Fig. 8 are shown, with active site half-cystine residues shown in bold. Note that the sequence for theBacillus alcalophilus ahpCis not complete at the 59 end.

117S. mutansperoxide reductase

Tab

le4.

Pai

rwis

eC

ompa

rison

sof

Ded

uced

Am

ino

Aci

dS

eque

nces

for

Bac

teria

lAhp

CH

omol

ogue

sa

Pro

xim

alA

hpF

hom

olog

uebN

oP

roxi

mal

Ahp

Fho

mol

ogue

Firm

icut

esc

Pro

teob

acte

riaF

irmic

utes

Bac

illus

/clo

strid

.gr

pLo

wG

1C

grm

pos

ge

aA

ctin

omyc

.

Str

ep

.m

uta

ns

Str

ep

.p

yog

.B

ac.

sub

.A

mp

hi.

xyl.

Sta

ph

.a

ure

us

En

ter.

fae

c.C

lost

r.p

ast

.S

alm

.ty

ph

.E

sch

.co

liP

seu

d.

pu

t.X

an

th.

cam

p.

Le

gio

np

ne

um

.H

elic

o.

pyl

.C

am

py.

jeju

n.

Ric

ket.

pro

w.

Myc

o.

tub

er.d

Co

ryn

e.

dip

hth

.A

qu

if.a

eo

l.S

yne

chsp

eci

es

Ch

lam

.p

ne

u.

Ch

lam

.tr

ach

.

S.

mu

tan

s10

088

.272

.669

.970

.554

.834

.362

.261

.363

.452

.236

.435

.034

.039

.635

.135

.648

.637

.738

.835

.0S

.p

yog

en

es

100

74.2

71.5

73.7

54.8

33.7

63.8

62.9

61.8

53.8

38.4

34.9

35.9

42.4

35.5

35.5

49.2

39.4

37.1

35.6

B.

sub

tilis

100

78.6

78.5

55.6

34.3

66.1

65.2

65.8

56.7

37.6

36.3

36.4

38.3

32.5

31.9

51.1

35.2

38.0

36.0

A.

xyla

nu

s10

076

.151

.334

.564

.563

.665

.259

.438

.438

.539

.338

.934

.236

.553

.836

.636

.937

.1S

.a

ure

us

100

50.3

34.9

66.1

65.2

62.6

55.7

39.0

36.0

33.3

35.8

32.1

32.6

47.8

36.5

34.3

35.0

E.

fae

calis

100

36.8

55.4

54.5

56.2

47.6

38.8

39.3

39.4

35.4

35.1

34.0

49.5

35.2

36.3

37.6

C.

pa

ste

uria

nu

m10

036

.236

.242

.141

.942

.540

.839

.145

.143

.345

.339

.253

.547

.139

.9S

.ty

ph

imu

riu

m10

098

.469

.257

.537

.334

.535

.638

.235

.137

.650

.036

.136

.533

.1E

.co

li10

067

.957

.238

.034

.135

.638

.935

.837

.650

.036

.135

.833

.7P

.p

utid

a10

065

.845

.043

.946

.141

.037

.741

.353

.844

.442

.540

.7X

.ca

mp

est

ris

100

36.8

35.0

36.4

36.1

33.8

38.5

46.2

37.9

36.3

37.1

L.

pn

eu

mo

ph

ilus

100

53.0

56.1

51.0

37.8

43.1

35.9

42.7

46.0

44.2

H.

pyl

ori

100

67.7

54.0

43.2

41.8

39.7

48.0

47.8

48.9

C.

jeju

ni

100

51.0

39.4

45.3

39.3

45.4

45.7

49.7

R.

pro

wa

zeki

i10

041

.144

.036

.351

.347

.943

.8M

.tu

be

rcu

losi

s10

067

.436

.344

.435

.942

.0C

.d

iph

the

ria

e10

038

.748

.041

.840

.7A

.a

eo

licu

s10

041

.734

.038

.0S

yne

ch.s

peci

es10

047

.944

.2C

.p

ne

um

on

iae

100

65.8

aS

yne

cho

cyst

isspe

cies

andA

qu

ifex

ae

olic

usb

oth

enco

dea

seco

ndA

hpC

hom

olog

uew

ithon

lyon

eof

the

two

cons

erve

dha

lf-cy

stin

ere

sidu

es(“

1-cy

spe

roxi

redo

xins

”)no

tsh

own

inth

ista

ble.

bA

llba

cter

iain

dica

ted

toha

vea

prox

imal

Ahp

Fho

mol

ogue

have

afu

ll-le

ngth

ah

pF

dow

nstr

eam

ofah

pC

exce

ptfo

rEn

tero

cocc

us

fae

calis,

whi

chha

sat

rxB

hom

olog

uedo

wns

trea

mofa

hp

C,an

dC

lost

rid

ium

pa

ste

uria

nu

m,w

hich

has

atrx

Bho

mol

ogue

upst

ream

ofah

pC

and

anin

terv

enin

gsm

allo

pen

read

ing

fram

e.c

Bas

edon

Gen

Ban

kcl

assi

ficat

ion.

dT

heM

yco

ba

cte

riu

mtu

be

rcu

losi

ssequ

ence

ishi

ghly

repr

esen

tativ

eof

sequ

ence

sal

sore

port

edfo

rM

.b

ovi

s,M

.sm

eg

ma

tis,

M.

avi

um,an

dM

.le

pra

e.

118 L. B. POOLE et al.

Since the original identification ofS. typhimuriumAhpC as a peroxidase in combination with AhpF, homo-logues of AhpC, also termed peroxiredoxins, have beenidentified in an extremely wide variety of organisms;multiple homologues appear to be expressed in mammalsand plants [6]. On the other hand, full-length AhpFhomologues are restricted to eubacteria according tosequence searches to date. Although the physiologicalsource of electrons for AhpC homologues from organ-isms lacking AhpF/Nox-1 proteins is not completelyclear, thioredoxin reductase and thioredoxin can fulfillthis function in some cases [29,30]. Table 4 clearlyillustrates the high degree of sequence identity amongbacterial AhpC proteins with dedicated AhpF/Nox-1 fla-voprotein reductases; interestingly, in several caseswhere AhpC homologues from organisms lacking fulllength AhpF homologues were tested for activity withS.typhimurium AhpF, no catalytic activity was detected(Entamoeba histolytica[30]; Mycobacterium bovis,Poole, Marcinkeviciene, and Blanchard, unpublisheddata;Helicobacter pylori, Baker and Poole, unpublisheddata). Bacterial AhpF proteins therefore exhibit a rela-tively high degree of specificity for their AhpC partners.

Comparisons of alkyl hydroperoxide reductase sys-tems fromS. mutansand S. typhimuriumhave, on theother hand, demonstrated the ability of these proteins tosubstitute for one another in peroxidase assays (Table 2);this result highlights the similarities between the gram-positive and gram-negative systems. The primary differ-ences between the two bacterial peroxidase systems arethe absolute specificity ofS. mutansNox-1 for NADHand the considerable difference in spectral features ob-served during reductive titrations of Nox-1 or AhpF. ForS. mutansNox-1 as well as theA. xylanusNADH oxi-dase [31], initial stages of the reductive titrations withNADH or dithionite involve almost no flavin reduction,suggesting that the redox potentials of the two disulfidecenters are higher and even farther separated from that ofthe flavin in these proteins relative toS. typhimuriumAhpF. Furthermore, stabilization of blue, neutralsemiquinone in the case of the gram-positive-derivedflavoproteins is less, at about 30%, relative to about 90%for S. typhimuriumAhpF. The generation of much morelong wavelength charge transfer species is also observedduring NADH titration of the gram positive flavopro-teins; this species is readily attributable to charge transferinteraction between FADH2 and NAD1, and one reasonfor its prominence in Nox-1 titrations with NADH is thegreater amount of reduced flavin relative to semiquinonegenerated during these titrations compared with AhpFtitrations. This species is formed quickly as detected inthe stopped flow spectrophotometer and may representan important catalytic species [18] (Li Calzi and Poole,unpublished data).

Although numerous in vitro characteristics of Nox-1/AhpC from S. mutansseem very similar to those ofS.typhimurium AhpF/AhpC, including catalytic parame-ters, an important role for this system inS. mutansinlowering the cytotoxicity of H2O2 or cumene hydroper-oxide has been difficult to demonstrate. InahpCF-defi-cient mutant strains ofS. typhimuriumor E. coli, diskinhibition assays demonstrated the hypersensitivity ofthese strains to cumene hydroperoxide [10]. Expressionof AhpF and AhpC proteins from introduced plasmidfully complemented and in fact augmented the resistanceof these bacteria toward this reagent. Overexpression ofthese proteins also had an “anti-mutator” effect inS.typhimuriumandE. coli DoxyRmutants with high levelsof spontaneous mutagenesis [32,33].S. mutansAhpCwith or without Nox-1 expressed from plasmid con-structs in anahpCF-deficient mutant strain ofE. coli wasalso able to complement the mutation and even enhancethe resistance toward cumene hydroperoxide [19].S.mutansstrains lacking one or both alkyl hydroperoxidereductase structural genes were not similarly sensitive tocumene hydroperoxide toxicity, however. Additional an-tioxidant protein(s) as yet unidentified inS. mutansmaymask the effects of deleting these genes [19].

Acknowledgements— This work was supported by grants from theJapan Society for the Promotion of Science (to L.B.P.) and MonbushoInternational Collaboration Travel Grant (to Y.K.), as well as NationalInstitutes of Health Grant RO1 GM50389 and Council for TobaccoResearch grant 4501 to L.B.P. and ISRP Grant 09044200 to Y.K. fromthe Ministry of Education, Science, Sports and Culture of Japan. Wethank Lois LaPrade and Chris Cecere for technical assistance and AlClaiborne for helpful discussions.

REFERENCES

[1] Hamada, S.; Slade, H. D. Biology, immunology, and cariogenic-ity of Streptococcus mutans. Microbiol. Rev.44:331–384; 1980.

[2] Higuchi, M. Effect of oxygen on the growth and mannitol me-tabolism ofStreptococcus mutans. J. Gen. Microbiol.130:1819–1826; 1984.

[3] Higuchi, M. Reduced nicotinamide adenine dinucleotide oxidaseinvolvement in defense against oxygen toxicity ofStreptococcusmutans. Oral Microbiol. Immun.7:309–314; 1992.

[4] Higuchi, M.; Shimada, M.; Yamamoto, Y.; Hayashi, T.; Koga, T.;Kamio, Y. Identification of two distinct NADH oxidases corre-sponding to H2O2-forming oxidase and H2O-forming oxidaseinduced inStreptococcus mutans. J. Gen. Microbiol.139:2343–2351; 1993.

[5] Higuchi, M.; Shimada, M.; Matsumoto, J.; Yamamoto, Y.; Rha-man, A.; Kamio, Y. Molecular cloning and sequence analysis ofthe gene encoding the H2O2-forming NADH oxidase fromStrep-tococcus mutans. Biosci. Biotech. Biochem.58:1603–1607; 1994.

[6] Chae, H. Z.; Robison, K.; Poole, L. B.; Church, G.; Storz, G.;Rhee, S. G. Cloning and sequencing of thiol-specific antioxidantfrom mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes.Proc. Natl. Acad. Sci. USA91:7017–7021; 1994.

[7] Poole, L. B. Flavin-dependent alkyl hydroperoxide reductasefrom Salmonella typhimurium. 2. Cystine disulfides involved incatalysis of peroxide reduction.Biochemistry35:65–75; 1996.

[8] Matsumoto, J.; Higuchi, M.; Shimada, M.; Yamamoto, Y.; Ka-

119S. mutansperoxide reductase

mio, Y. Molecular cloning and sequence analysis of the geneencoding the H2O-forming NADH oxidase fromStreptococcusmutans. Biosci. Biotech. Biochem.60:39–43; 1996.

[9] Ross, R. P.; Claiborne, A. Molecular cloning and analysis of thegene encoding the NADH oxidase fromStreptococcus faecalis10C1. Comparison with NADH peroxidase and the flavoproteindisulfide reductases.J. Mol. Biol. 227:658–671; 1992.

[10] Storz, G.; Jacobson, F. S.; Tartaglia, L. A.; Morgan, R. W.;Silveira, L. A.; Ames, B. N. An alkyl hydroperoxide reductaseinduced by oxidative stress inSalmonella typhimuriumandEsch-erichia coli: genetic characterization and cloning ofahp. J Bac-teriol 171:2049–2055; 1989.

[11] Li Calzi, M.; Poole, L. B. Requirement for the two AhpF cystinedisulfide centers in catalysis of peroxide reduction by alkyl hy-droperoxide reductase.Biochemistry36:13357–13364; 1997.

[12] Poole, L. B. TheSalmonella typhimuriumalkyl hydroperoxidereductase enzyme system. In: Stevenson, K. J.; Massey, V.;Williams, C. H., Jr., eds.Flavins and Flavoproteins 1996. Cal-gary, Alberta, Canada: University of Calgary Press; 1997:751–760.

[13] Poole, L. B.; Shimada, M.; Higuchi, M. NADH oxidase-1 and asecond component encoded upstream ofnox1comprise an alkylhydroperoxide reductase system inStreptococcus mutans. In:Stevenson, K. J.; Massey, V.; Williams, C. H., Jr., eds.Flavinsand Flavoproteins 1996. Calgary, Alberta, Canada: University ofCalgary Press; 1997:769–772.

[14] Niimura, Y.; Poole, L. B.; Massey, V.Amphibacillus xylanusNADH oxidase andSalmonella typhimuriumalkyl hydroperoxidereductase flavoprotein component show extremely high scaveng-ing activity for both alkyl hydroperoxide and hydrogen peroxidein the presence ofS. typhimuriumalkyl hydroperoxide reductase22-kDa protein component.J. Biol. Chem.269:25645–25650;1995.

[15] Niimura, Y.; Ohnishi, K.; Nishiyama, Y.; Kawasaki, S.; Miyaji,T.; Suzuki, H.; Nishino, T.; Massey, V.Amphibacillus xylanusNADH oxidase/alkyl hydroperoxide reductase flavoprotein. In:Stevenson, K. J.; Massey, V.; Williams, C.H., Jr., eds.Flavinsand Flavoproteins 1996. Calgary, Alberta, Canada: University ofCalgary Press; 1997:741–750.

[16] Koyama, N.; Koitabashi, T.; Niimura, Y.; Massey, V. Peroxidereductase activity of NADH dehydrogenase of an alkaliphilicBacillus in the presence of a 22-kDa protein component fromAmphibacillus xylanus. Biochem. Biophys. Res. Commun.247:659–662; 1998.

[17] Loprasert, S.; Atichartpongkun, S.; Whangsuk, W.; Mongkolsuk,S. Isolation and analysis of theXanthomonasalkyl hydroperoxidereductase gene and the peroxide sensor regulator genesahpCandahpF-oxyR-orfX. J. Bacteriol.179:3944–3949; 1997.

[18] Niimura, Y.; Massey, V. Reaction mechanism ofAmphibacillusxylanusNADH oxidase/alkyl hydroperoxide reductase flavopro-tein. J. Biol. Chem.271:30459–30464; 1996.

[19] Higuchi, M.; Yamamoto, Y.; Poole, L. B.; Shimada, M.; Sato, Y.;Takahashi, N.; Kamio, Y. Functions for two types of NADHoxidases in energy metabolism and oxidative stress ofStrepto-coccus mutans. J. Bacteriol.181:5940–5947; 1999.

[20] Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. D.; Seid-man, J. G.; Smith, J. A.; Struhl, K.Short protocols in molecularbiology. New York: John Wiley and Sons; 1992.

[21] Poole, L. B.; Ellis, H. R. Flavin-dependent alkyl hydroperoxidereductase fromSalmonella typhimurium. 1. Purification and en-zymatic activities of overexpressed AhpF and AhpC proteins.Biochemistry35:56–64; 1996.

[22] Jacobson, F. S.; Morgan, R. W.; Christman, M. F.; Ames, B. N.An alkyl hydroperoxide reductase fromSalmonella typhimuriuminvolved in the defense of DNA against oxidative damage. Puri-fication and properties.J. Biol. Chem.264:1488–1496; 1989.

[23] Ellis, H. R.; Poole, L. B. Roles for the two cysteine residues ofAhpC in catalysis of peroxide reduction by alkyl hydroperoxidereductase from Salmonella typhimurium. Biochemistry36:13349–13356; 1997.

[24] Shine, J.; Dalgarno, L. Determinant of cistron specificity in bac-terial ribosomes.Nature254:34–38; 1975.

[25] Rosenberg, M.; Court, D. Regulatory sequences involved in thepromotion and termination of RNA transcription.Ann. Rev. Gen.13:319–353; 1975.

[26] Niimura, Y.; Ohnishi, K.; Yarita, Y.; Hidaka, M.; Masaki, H.;Uchimura, T.; Suzuki, H.; Kozaki, M.; Uozumi, T. A flavoproteinfunctional as NADH oxidase fromAmphibacillus xylanusEp01:purification and characterization of the enzyme and structuralanalysis of its gene.J. Bacteriol.175:7945–7950; 1993.

[27] Massey, B.; Palmer, G. On the existence of spectrally distinctclasses of flavoprotein semiquinones. A new method for thequantitative production of flavoprotein semiquinones.Biochemis-try 5:3181–3189; 1966.

[28] Ellis, H. R.; Poole, L. B. Novel application of 7-chloro-4-nitro-benzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in theAhpC component of alkyl hydroperoxide reductase.Biochemistry36:15013–15018; 1997.

[29] Chae, H. Z.; Chung, S. J.; Rhee, S. G. Thioredoxin-dependentperoxide reductase from yeast.J. Biol. Chem.269:27670–27678;1994.

[30] Poole, L. B.; Chae, H.-Z.; Flores, B. M.; Reed, S. L.; Rhee, S. G.;Torian, B. E. Peroxidase activity of a TSA-like antioxidant pro-tein from a pathogenic amoeba.Free Radic. Biol. Med.23:955–959; 1997.

[31] Ohnishi, K.; Niimura, Y.; Yokoyama, K.; Hidaka, M.; Masaki,H.; Uchimura, T.; Suzuki, H.; Uozumi, T.; Kozaki, M.; Koma-gata, K.; Nishino, T. Purification and analysis of a flavoproteinfunctional as NADH oxidase fromAmphibacillus xylanusover-expressed inEscherichia coli. J. Biol. Chem.269:31418–31423;1994.

[32] Storz, G.; Christman, M. F.; Sies, H.; Ames, B. N. Spontaneousmutagenesis and oxidative damage to DNA inSalmonella typhi-murium. Proc. Natl. Acad. Sci. USA84:8917–8921; 1987.

[33] Greenberg, J. T.; Demple, B. Overproduction of peroxide-scav-enging enzymes inEscherichia colisuppresses spontaneous mu-tagenesis and sensitivity to redox-cycling agents inoxyR- mu-tants.EMBO J.7:2611–2617; 1988.

120 L. B. POOLE et al.