Reactive oxygen species are harmful to the cell by oxidizing various cell components, such as lipids, nucleic acids, and proteins. Among the amino acids, methionine (Met) residues are known to be particularly susceptible to oxidative stress and are easily oxidized to methionine sulfoxides (MetO). Methionine sulfoxide reductase (Msr) is an enzyme that repairs the oxidized methionine and catalyzes the thioredoxin-dependent reduction of MetO to Met (6). The oxidation of Met results in the formation of two asymmetric molecules, Met-S-O and Met-R-O. Each MetO is reduced by a specific and structurally distinct enzyme: Met-S-O is reduced by MsrA, and Met-R-O is reduced by MsrB. Msr proteins are considered to play vital roles in maintaining the intracellular redox balance and in the repair of oxidized proteins (5, 9). In mammals, defects in the function of Msr have been reported to result in neurological disorders and, in some cases, a decrease in life span (30, 31). Although structurally distinct, MsrA and MsrB catalyze the reduction of MetO with basically similar mechanisms (2, 4, 20, 33). A cysteine residue, designated CysA, acts as a nucleophile that attacks the oxidized sulfur atom of MetO. A tetrahedral transition state is formed, followed by a rearrangement that releases the repaired Met, and this results in the formation of a sulfenic acid intermediate on the CysA side chain. A second cysteine residue, CysB, then attacks the oxidized CysA and, along with the release of a water molecule, forms a disulfide bond with CysA. Other cysteine residues may participate in the steps that follow, but the enzyme eventually is reduced in a thioredoxin-dependent manner, completing the reaction. MsrA proteins can be classified into three main groups by the number and positions of cysteine residues that are proposed to be involved in the catalytic mechanism (20). The first group (MsrAI) utilizes three Cys residues in catalysis, and the nucleophilic CysA residue is conserved in a GCFWG motif. The CysB residue is conserved in a GYCG sequence. A third Cys residue (CysC) is present in MsrAI and resides downstream of a glycine-rich sequence. The second group of MsrA proteins (MsrAII) utilizes only two Cys residues, basically corresponding to CysA and CysB of MsrAI enzymes. The third group of enzymes (MsrAIII) harbors both CysA and CysB residues in a single GCFWC motif. The MsrB proteins are classified into two groups by the presence (form I) or absence (form II) of two CxxC motifs that have been shown to participate in binding to a divalent zinc cation in the MsrB from Drosophila melanogaster (26). Although mutations in any one of these four Cys residues leads to a nonactive protein, this cluster seems to play a structural role in the enzyme. All MsrB proteins from eukaryotes and archaea are form I enzymes, while bacteria harbor either form I or form II, depending on the species. Consistent with their important roles in dealing with oxygen or oxidative stress, Msr proteins are widely distributed in nature and can be found in all three domains of life, Eucarya, Bacteria, and Archaea. Msr homologs are present in almost all mesophile genomes sequenced thus far. However, almost all hyperthermophiles from the Bacteria and the Archaea do not harbor Msr genes. The only exceptions are the MsrA homolog in Sulfolobus solfataricus (43) and the MsrA-MsrB fusion homolog in Thermococcus kodakaraensis (designated MsrABTk) (11) (Table (Table1).1). The presence of Msr in T. kodakaraensis was particularly intriguing, as Msr homologs are not present in any of the genome sequences from Pyrococcus furiosus (37), “Pyrococcus abyssi” (7), and Pyrococcus horikoshii (22), which are very closely related to T. kodakaraensis. TABLE 1. The presence of Msr homologs in various archaea along with their growth temperatures The genera Pyrococcus and Thermococcus both belong to the family Thermococcales and consist of heterotrophic, sulfur-reducing anaerobes that share common metabolism and energy-generating mechanisms (1, 11, 48). The major distinction between Thermococcus and Pyrococcus is in their growth temperatures; the former has optimal growth temperatures between 75 and 93°C, while those of the latter range from 95 to 103°C (1, 18). Although the Pyrococcus spp. have received relatively more attention in terms of biochemical and genome research, environmental studies have indicated that Thermococcus seems to be by far the more predominant genus distributed throughout the hydrothermal environments on our planet (16, 18, 35). Thermococcus also seems to be much more diverse (18), including members that can grow at alkaline pH (T. alcaliphilus) (23), extremely low salinity (T. waiotapuensis) (13), and at temperatures as low as 40°C (T. sibiricus) (29). In the natural environment, a decrease in temperature usually brings about an increase in dissolved oxygen (DO) concentration. Thus, the Thermococcus spp., which generally grow at lower temperature ranges, may harbor additional defense mechanisms against oxygen that are not present in Pyrococcus spp. As an Msr gene is present on the T. kodakaraensis genome but is absent from the three Pyrococcus genomes, there is a possibility that Msr represents one of these additional mechanisms. In this study, we have examined the biochemical properties of MsrABTk and its presence in T. kodakaraensis under various growth conditions.