sensitive to O2, the cycle commonly functions in anaerobic and microaerobic organisms [18–21]. However, H. thermophilus prefer- entially grows under aerobic conditions, even at O2 concentrations as high as 40%; thus, elucidation of the ROS detoxification system in this microorganism is of considerable interest. In 2008, the whole-genome sequence of H. thermophilus was completed [22], and a gene encoding a single-domain Rbr-like protein was identified in the genome (Fig. S2). In the present study, we investigated the potential role of this Rbr-like protein as an antioxidant in vitro and in vivo. Furthermore, the structural significance and phylogeny of this protein was evaluated by comparison of the primary and tertiary structure with numerous Rbr-like proteins. Here, we show that this Rbr-like protein acts as an NADPH-FNR-dependent peroxidase and is an essential antioxidant for H. thermophilus in aerobic environments. We propose naming this protein ferriperoxin (Fpx) based on the observed structural and functional features. Results Heterologous expression and purification of Fpx Recombinant Fpx was expressed in E. coli under control of the T7 promoter in the pET vector. The cell-free extract (CFE) of the recombinant E. coli strain exhibited a yellow-greenish color, suggesting that Fpx was expressed as a holoprotein, as Fpx was predicted to possess a diiron domain that is known to have absorption in the visible range (<375 nm) [1]. A yellow-green protein was purified to homogeneity under aerobic conditions at room temperature (RT) by a combination of heat-treatment and repeated anion-exchange chromatography (Fig. S3). The N- terminal amino acid sequence of the purified protein was determined to be MKSLAGTKTL, which was identical to the deduced amino acid sequence of Fpx, indicating that the recombinant protein was derived from the fpx gene. The purified Fpx appeared as a single 16-kDa band on SDS-PAGE (Fig. S3A), closely corresponding to the calculated molecular mass of monomeric Fpx based on the sequence of fpx (15.7 kDa). To determine the subunit composition of recombinant Fpx, gel filtration was performed with purified Fpx (Fig. S3B). The molecular mass of recombinant Fpx was estimated to be 29.4 kDa, suggesting that Fpx exists as a dimeric protein, similar to typical two-domain Rbr, SulE, and rubperoxin (Rpr) isolated from other organisms [1,12,23,24]. UV-visible spectrum of Fpx Purified Fpx was yellow-green in the air-oxidized state. The UV-visible spectrum of air-oxidized Fpx (Fpxox) showed a broad absorption and two peaks at 330 and 370 nm, respectively (Fig. 1A), in contrast to that of typical two-domain Rbrs, which are reported to possess peaks at 375, 500, and 580 nm [1,25]. It has been demonstrated that the 375-nm peak corresponds to the N- terminal diiron domain, and the 500- and 580-nm peaks correspond to the C-terminal Rdx-domain in two-domain Rbrs [1]. The absence of peaks at 500 or 580 nm in the spectrum of Fpxox coincided with the absence of the Rdx domain. The characteristic peak of Fpxox at 370 nm disappeared after exposure of Fpx to the strong reductant dithionite (Fig. 1A), similar to the findings of typical two-domain Rbrs [9,25]. This result indicates that Fpx has both oxidized and reduced states, suggesting that Fpx functions as a redox protein. With regard to the potential physiological redox partners of Rbr homologs, two classes of candidate enzymes have been reported; one is Rdx/Rdx reductase and the second is FNR [6,9,25]. H. thermophilus possesses FNR, but homologs of Rdx or Rdx reductase An Essential Antioxidant in H. thermophilus have not been identified. Thus, we examined the capability of FNR to function as the redox partner of Fpx. Although no reduction of Fpxox was observed by the addition of NADPH alone to a purified protein solution (Fig. 1Bb), Fpxox was reduced when both NADPH and FNR were added, indicating that FNR was effective for mediating electron transfer from NADPH to Fpx (Fig. 1Bc). In addition, by exposure of Fpxred to H2O2, the solution showed the spectrum same as oxidized Fpx, indicating that reduced Fpx was reoxidized by H2O2. This observation suggests that Fpx can function as an NADPH-FNR-dependent peroxidase. Enzyme assays Rbr homologs from several organisms have been reported to possess peroxidase activity by obtaining reducing power from NAD(P)H via redox partners [6,9,25,26]. To determine whether Fpx displays NADPH-dependent peroxidase activity, its activities toward H2O2, t-butyl hydroperoxide (t-BOOH), and cumene hydroperoxide (CMOOH) were measured using FNR as a redox partner. Fpx showed peroxidase activity when Fpx, FNR, and NADPH were present in the reaction mixture, whereas no activity was observed when any of one of three was absent (data not shown). The optimal pH for peroxidase activity of Fpx (<pH 6.0) was lower than that of other Rbr homologs (pH 7.0 to 8.0) [6,9,25,26] (Fig. S4). The initial rate of hydroperoxide consumption by Fpx was directly measured at various concentrations of H2O2, t-BOOH, and CMOOH in the presence of NADPH and FNR. The analysis of the resulting Michaelis-Menten plots showed that the Km values of Fpx for H2O2, t-BOOH, and CMOOH were 1.660.2, 1.360.3, and 1.160.2 mM, respectively, and the Vmax values were 2761, 2462, and 1161 mmol/min/mg protein, respectively (Fig. 2). The Vmax/Km values of Fpx for H2O2, t-BOOH, and CMOOH were calculated to be 17, 18, and 10 mmol/min/mg protein, respectively. The reactivity of Fpx with superoxide produced by the xanthine/xanthine oxidase system was evaluated using a SOD Assay Kit-WST. SOD activity of Fpx was determined to be 0.10 U mg protein21. This activity is very low compared to the reported values for bovine Cu, ZnSOD and E. coli Mn-SOD (2000-4000 U mg protein21) [27,28]. Hence, it is unlikely that Fpx is involved in superoxide anion detoxification systems. The reactivity of Fpx with O2 was also analyzed. Reduced Fpx (48 mM) was slowly oxidized by dissolved O2 (213 mM) at an initial rate of reduction that corresponded to 0.40 nmol O2 min21 mg protein21. This calculated rate is markedly lower than to the rates of O2 reduction by terminal oxidases isolated from other organisms (ranging from 40–90 nmol O2 min21 mg protein21) [29,30], indicating that Fpx is not involved in O2-reduction systems. Physiology of an fpx gene disruptant To investigate the in vivo antioxidant function of Fpx, the fpx gene disruptant (Dfpx) was constructed by a homologous re- combination method with minor alterations [31]. The disruption of the fpx gene was confirmed by PCR. As expected, the PCR fragment was 1.1 kb longer than that of the wild-type (WT) strain, indicating that the disruption of the fpx gene by insertion of the highly thermostable kanamycin nucleotidiyltransferase (htk) gene gene was successful. To test the sensitivity of Dfpx toward oxidative stress, the mutant and WT strains were cultured under aerobic (10% O2), anaerobic (0% O2, denitrification), and microaerobic (2% O2) conditions. Growth curves of the Dfpx and WT strains are shown in Fig. 3. Although the growth rate of WT H. thermophilus was highest under aerobic conditions, Dfpx was unable to grow in the presence of 10% O2, indicating the indispensability of Fpx for PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34825