Modeling of three-dimensional structure Three-dimensional structure of Fpx was generated using the SWISS-MODEL automated homology modeling server (http:// swissmodel.expasy.org/) Construction of phylogenetic tree Based on 507 amino acid sequences obtained from the NCBI protein data-base, the phylogenetic tree of proteins annotated as Rbr or Rbr-like proteins were constructed using the maximum- likelihood method. The 54 sequences that showed markedly high homology to ferritin were used as out-groups. The branching point of ferritin group was defined as the root of the phylogenetic tree. Detailed information of the sequences used in the analysis is shown in Table S1. Although the reliability of the order of intergroup divergences between groups III, IV, and V were low, those of the other intergroup divergences were confirmed to be high (bootstrap value .70%) with other phylogenetic trees. Other methods UV-visible spectra were measured on a Beckman DU 7400 spectrophotometer. The reduction and oxidation of Fpx were observed by monitoring its spectral changes under anaerobic conditions using 1-cm-pass-length anaerobic quartz cuvettes. Chemical reduction of Fpx was achieved by the addition of 5 mM sodium dithionite to a solution containing 100 mM Fpx and 20 mM Tris-HCl [pH 8.0] (buffer A) under anaerobic conditions, which were generated by replacing the head space of cuvettes with argon. Partial reduction of Fpx by FNR/NADPH was achieved by the addition of 50 mM NADPH and 0.13 mM FNR to bufferA containing 100 mM Fpx, and incubation for 10 min at 70uC. Supporting Information Figure S1 Phylogenetic tree of 1- and 2-domain types of rubrerythrin-like proteins based on amino acid se- quence. One hundred amino acid sequences were obtained by BLAST search using the amino acid sequence of Hydrogenobacter thermophilus Fpx (Hth). The 100 sequences and three additional amino acid sequences of Rbrs (Desulfovibrio vulgaris, Dvu; Pyrococcus furiosus, Pfu; and Anabaena sp. PCC7120, Ana), which have been investigated and reported to date, were used for the phylogenetic tree. The tree was constructed by using maximum-likelihood method. The group of the 1-domain type protein consists of the sequences from organisms including: 5 a-proteobacteria (a), 26 b- proteobacteria (b), 6 c-proteobacteria (c), 6 d-proteobacteria (d), 6 actinobacteria, 4 Aquificae, 1 unclassified bacteria, 10 Thermo- plotei including Sulfolobus tokodaii (Sto) and 1 Eukaryote. This group also includes 56 aerobic, 4 microaerobic (closed circle) and 1 anaerobic (closed triangle) (and 6 unknown) organisms. The group References 1. LeGall J, Prickril BC, Moura I, Xavier AV, Moura JJ, et al. (1988) Isolation and characterization of rubrerythrin, a non-heme iron protein from Desulfovibrio vulgaris that contains rubredoxin centers and a hemerythrin-like binuclear iron cluster. Biochemistry 27: 1636–1642. 2. Lehmann Y, Meile L, Teuber M (1996) Rubrerythrin from Clostridium perfringens: cloning of the gene, purification of the protein, and characterization of its superoxide dismutase function. J Bacteriol 178: 7152–7158. 3. Alban PS, Popham DL, Rippere KE, Krieg NR (1998) Identification of a gene for a rubrerythrin/nigerythrin-like protein in Spirillum volutans by using amino acid sequence data from mass spectrometry and NH2-terminal sequencing. J Appl Microbiol 85: 875–882. 4. Das A, Coulter ED, Kurtz DM Jr., Ljungdahl LG (2001) Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubre- doxin oxidoreductase- rubredoxin and rubrerythrin-type A flavoprotein- high- molecular-weight rubredoxin. J Bacteriol 183: 1560–1567. An Essential Antioxidant in H. thermophilus of the 2-domain type protein consists of the sequences from 34 anaerobic organisms. (TIF) Figure S2 Amino acid sequences of Fpx, SulE, and two Rbr proteins. Residues liganded to metal centers and those conserved among all four sequences are underlined and shown in bold, respectively. Predicted secondary structure of Fpx is shown above the sequences; dotted lines and an arrow represent the a- helix and loop, respectively. Hth, Hydrogenobacter thermophilus Fpx; Sto, Sulfolobus tokodaii SulE; Pfu, Pyrococcus furiosus Rbr; and Dvu, Desulfovibrio vulgaris Rbr. (TIF) Figure S3 Characterization of recombinant Fpx, a ru- brerythrin-like protein from H. thermophilus. A. SDS- PAGE analysis of the purified recombinant Fpx on a 13% acrylamide gel. Lane 1, 11 mg of purified Fpx, which had an apparent molecular mass of 16 kDa. B. Elution profiles of Fpx and selected molecular standards. Molecular mass of native subunit composition of Fpx was determined by gel filtration using a Superose 6 HR column equilibrated with 20 mM Tris-F|HCl and 150 mM NaCl [pH 8.0]. The profiles of the standards are as follows: thyroglobulin, 670 kDa, 12.48 ml; c-globulin, 158 kDa, 14.24 ml; ovalbumin, 44 kDa, 16.01 ml; myoglobin, 17 kDa, 17.0 ml; and vitamin B12, 1.35 kDa, 19.83 ml (from top to bottom). The molecular mass of Fpx was calculated to be 29.4 kDa. (TIF) Figure S4 The pH-dependent changes of peroxidase activity of Fpx. In order to determine the optimal pH for peroxidase activity of Fpx, pH-dependency of peroxidase activity of Fpx was analyzed. Peroxidase assays were routinely carried out anaerobically in a 200-ml reaction mixture containing 0.32 mM Fpx, 0.0074 mM FNR, 0.5 mM NADPH, 100 mM hydroper- oxides (H2O2 or t-butyl hydroperoxide (t-BOOH)) and 50 mM sodium phosphate [pH 6.0], at 50uC for 20 min. The reaction was started by addition of NADPH after 1.5-min preincubation. The determination of the concentrations of hydroperoxides was performed by according to the report of Wolf et al. with minor modification. A, the result using H2O2; B, t-BOOH. (TIF) Table S1 The list of organisms used for the phyloge- netic tree of Rbr. (PDF) Author Contributions Conceived and designed the experiments: YS MK MI HA YI. Performed the experiments: YS MK SF. Analyzed the data: YS MK SF TW MI HA. Wrote the paper: YS MK SF. 5. Sztukowska M, Bugno M, Potempa J, Travis J, Kurtz DM Jr. (2002) Role of rubrerythrin in the oxidative stress response of Porphyromonas gingivalis. Mol Microbiol 44: 479–488. 6. Weinberg MV, Jenney FE Jr., Cui X, Adams MW (2004) Rubrerythrin from the hyperthermophilic archaeon Pyrococcus furiosus is a rubredoxin-dependent, iron- containing peroxidase. J Bacteriol 186: 7888–7895. 7. Pierik AJ, Wolbert RB, Portier GL, Verhagen MF, Hagen WR (1993) Nigerythrin and rubrerythrin from Desulfovibrio vulgaris each contain two mononuclear iron centers and two dinuclear iron clusters. Eur J Biochem 212: 237–245. 8. Bonomi F, Kurtz DM, Cui XY (1996) Ferroxidase activity of recombinant Desulfovibrio vulgaris rubrerythrin. Journal of Biological Inorganic Chemistry 1: 67–72. PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e34825