An Essential Antioxidant in H. thermophilus Figure 1. Spectrum analyses of Fpx. A. Absorption spectra of 100 mM air-oxidized (curve a) and sodium dithionite (5 mM)-reduced (curve b) Fpx in 20 mM Tris-HCl [pH 8.0] (buffer A). B. Partial reduction of Fpx by FNR/NADPH. Curve a shows the spectrum of oxidized Fpx (100 mM) in buffer A. Oxidized Fpx in buffer A was incubated with 50 mM NADPH (curve b) or with both 50 mM NADPH and 0.13 mM FNR (curve c) for 10 min at 70uC. Reduced Fpx was reoxidized by H2O2 (curve a). doi:10.1371/journal.pone.0034825.g001 the aerobic growth of H. thermophilus (Fig. 3A). In contrast, the growth profiles of Dfpx was comparable to that of WT under both anaerobic and microaerobic conditions. To reveal whether the inhibition of the aerobic growth of Dfpx resulted from the defect in the ROS detoxification system, the abilities of WT and Dfpx for survival in the presence of ROS were observed (Fig. 4). WT strain was capable of growth in the presence of low concentrations of ROS (e.g. 1–10 mM paraquat and 10– 50 mM H2O2). In contrast, even low concentrations of ROS showed bactericidal or inhibition effects on the mutant, strongly indicating a specific defect of the defense system against oxidative stress in Dfpx. Collectively these results suggest that Fpx acts as an important antioxidant in vivo, and is essential for the aerobic growth of H. thermophilus. Structural modeling and phylogenetic analysis By homology modeling, the three-dimensional structure of Fpx was predicted using the crystal structure of SulE as a template (Fig. 5). The primary structure of Fpx shares 69% identity with that of SulE. The homology modeling also predicted that domain- swapping, in which a secondary or tertiary element of a monomeric protein is replaced by the same element of another protein, was predicted to occur in Fpx, similar to that reported for SulE [23]. Based on 507 amino acid sequences, the phylogenetic tree of proteins annotated as Rbr or Rbr-like proteins were constructed. The 507 sequences were divided into eight groups including out- group. Groups I, II, and III are composed of Rprs and the single- domain Rbr-like proteins, including Fpx, while typical two- domain Rbrs are distributed among groups IV to VII. Discussion In this study, we demonstrated that the Rbr-like protein Fpx of H. thermophilus functions as a peroxidase that couples with the NADPH- FNR system. Fpx showed specific peroxidase activity toward both H2O2 and organic hydroperoxides. It was unexpected that the values of Km and Vmax for t-BOOH and CMOOH were comparable to those for H2O2, because Rpr is reported to have almost no alkyl hydroperoxide reductase activity [12], although the activities of the other characterized Rbr homologs to date have not been reported. Thus, the multiple reactivities toward hydroperoxides appear to be a characteristic feature of Fpx. We also demonstrated by gene disruption that Fpx acts as a vital antioxidant in vivo. By the Figure 2. NADPH-FNR-dependent peroxidase activity of Fpx. Michaelis-Menten plots of the initial rates of removal of H2O2 (A), t-BOOH (B), or cumene hydroperoxide (CMOOH) (C) by Fpx versus the concentration of the respective peroxides. The initial rates of peroxide removal were determined directly by measuring the amount of peroxides reduced by Fpx in the presence of FNR and NADPH for 20 min at 50uC. One unit of peroxidase activity is defined as the amount of activity that can reduce 1 mmol of peroxide per min. Each value was calculated based on the measurements of triplicate samples. Error bars show the standard deviations of each triplicate values. doi:10.1371/journal.pone.0034825.g002 PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34825