A Novel Enzymatic System against Oxidative Stress in the Thermophilic Hydrogen-Oxidizing Bacterium Hydrogenobacter thermophilus Yuya Sato1, Masafumi Kameya2, Shinya Fushinobu1, Takayoshi Wakagi1, Hiroyuki Arai1, Masaharu Ishii1*, Yasuo Igarashi1 1 Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan, 2 Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Imizu, Toyama, Japan Abstract Rubrerythrin (Rbr) is a non-heme iron protein composed of two distinctive domains and functions as a peroxidase in anaerobic organisms. A novel Rbr-like protein, ferriperoxin (Fpx), was identified in Hydrogenobacter thermophilus and was found not to possess the rubredoxin-like domain that is present in typical Rbrs. Although this protein is widely distributed among aerobic organisms, its function remains unknown. In this study, Fpx exhibited ferredoxin:NADPH oxidoreductase (FNR)-dependent peroxidase activity and reduced both hydrogen peroxide (H2O2) and organic hydroperoxide in the presence of NADPH and FNR as electron donors. The calculated Km and Vmax values of Fpx for organic hydroperoxides were comparable to that for H2O2, demonstrating a multiple reactivity of Fpx towards hydroperoxides. An fpx gene disruptant was unable to grow under aerobic conditions, whereas its growth profiles were comparable to those of the wild-type strain under anaerobic and microaerobic conditions, clearly indicating the indispensability of Fpx as an antioxidant of H. thermophilus in aerobic environments. Structural analysis suggested that domain-swapping occurs in Fpx, and this domain- swapped structure is well conserved among thermophiles, implying the importance of structural stability of domain- swapped conformation for thermal environments. In addition, Fpx was located on a deep branch of the phylogenetic tree of Rbr and Rbr-like proteins. This finding, taken together with the wide distribution of Fpx among Bacteria and Archaea, suggests that Fpx is an ancestral type of Rbr homolog that functions as an essential antioxidant and may be part of an ancestral peroxide-detoxification system. Citation: Sato Y, Kameya M, Fushinobu S, Wakagi T, Arai H, et al. (2012) A Novel Enzymatic System against Oxidative Stress in the Thermophilic Hydrogen- Oxidizing Bacterium Hydrogenobacter thermophilus. PLoS ONE 7(4): e34825. doi:10.1371/journal.pone.0034825 Editor: Vasu D. Appanna, Laurentian University, Canada Received November 15, 2011; Accepted March 6, 2012; Published April 2, 2012 Copyright: 2012 Sato et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Grant-in-Aid for Scientific Research (A) from Japan Society for the Promotion of Science KAKENHI (21248010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: amishii@mail.ecc.u-tokyo.ac.jp Introduction Molecular oxygen (O2) is the most abundant oxidant existing in the earth’s surface, an environment in which numerous organisms are distributed. As the aerobic respiration of O2 permits high energy yields, many organisms have evolved the ability to utilize O2. However, the utilization of O2 is also associated with the risk of cellular damage from reactive oxygen species (ROS) that are derived from O2-related metabolism. ROS have high reactivity towards numerous cellular molecules, including nucleic acids and proteins, leading to irreversible damage and even cell death. To detoxify ROS, cells are equipped with various systems, such as superoxide reductase, catalase, hydrogen peroxide reductase, and organic peroxide reductase. Rbr is a non-heme iron homodimeric protein containing a ferritin-like diiron domain at the N-terminus and a rubredoxin (Rdx)-like domain at the C-terminus. Rbr was originally identified in the anaerobic sulfate reducing bacterium Desulfovibrio vulgaris and is widely distributed in anaerobic bacteria and archaea [1–6]. Although Rbr has been reported to possess pyrophosphatase [7], ferroxidase [8], and superoxide dismutase (SOD) [2] activities, recent evidence suggests that it functions as a H2O2 reductase [6,9–11] to protect cells against oxidative stress. Several types of Rbr-like proteins have been identified to date [7,12,13]. Among them, sulerythrin (SulE), which is composed of only a ferritin-like domain, was identified in the aerobic archaea Sulfolobus tokodaii, however, its function remains unknown [13]. By BLAST analysis, the genes encoding this single-domain-type Rbr- like protein were found to be widely distributed among aerobic and facultative anaerobic bacteria, and archaea, whereas typical Rbr genes are distributed among anaerobes (Fig. S1). Further, it has been reported that ferritin-like diiron domain catalyzes 2-electron reduction of H2O2, and the reaction mechanism has been proposed [14]. Based on the observed distribution and reported ability to reduce H2O2, it is possible that single-domain Rbr-like proteins play a role in oxidative-stress defense systems in aerobic environments. Hydrogenobacter thermophilus is a thermophilic, hydrogen-oxidizing bacterium [15] that is located at the deepest branch of the domain Bacteria based on the 16S rRNA phylogeny [16]. H. thermophilus is a chemolithoautotroph that assimilates carbon dioxide (CO2) as the sole carbon source via the reductive tricarboxylic acid (RTCA) cycle [17]. As several key enzymes of the RTCA cycle are highly PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34825