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The peroxiredoxins (PRDXs) define a superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high-molecular-weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 Å resolution. Although A. marina PRDX6 was cloned during the search of annelid homologs of mammalian 1-Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2-Cys PRDX subfamily. The protein is composed of two distinct domains: a C-terminal domain and an N-terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild-type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid-shaped decamers formed by dimer association and observed in most typical 2-Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2-Cys PRDXs without any formation of toroid-shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide-dependent cell signaling rather than a molecular chaperone.

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T his authors' personal cop y m ay not be publicly o r syste m a tica lly copied or distributed, o r posted on the O pen W eb, exce pt w ith w ritten perm ission o f th e co p yrig h t holder(s). It m ay be distributed to interested individuals on request. ABSTRACT: The hydrotherm al Pompeii worm Alvinella pom pejana lives very close to the walls of black smokers and is therefore exposed to high-tem perature venting fluid containing high con centrations of sulphides and metals. The highly aerobic m etabolism of these annelids, together with these extrem e physico-chemical conditions, theoretically accelerates redox processes in and around the worm, potentially increasing oxidative threat by reactive oxygen species (ROS). This prom pted us to analyse activity of antioxidant enzymes in A. pom pejana tissues and investigate w hether they are adjusted to the endogenous production of ROS by oxidative phosphorylations and/or to the environm ental conditions. This was investigated by com paring antioxidant and m etabolic enzyme activities in gills, head, body wall, pygidium and guts of A. pom pejana col lected at different vent sites of the East Pacific Rise. The antioxidant defence arsenal of A. p o m p e jana is peculiar, showing very low catalase (CAT) activity and very high superoxide dism utase (SOD) activity in most tissues. It is very likely that CAT is not expressed in A. pom pejana, as this haem ic enzyme could be inhibited by the high sulphide concentrations prevailing in the worm's environm ent. A. pom pejana does not com pensate for the low hydrogen peroxide scavenging activity of CAT by higher glutathione peroxidase (GPX) activity levels. This latter enzyme corre lates well with cytochrome c oxidase and citrate synthase in most tissues, suggesting that oxida tive m etabolism represents the m ain source of peroxides m anaged by GPX. On the contrary, SOD shows no correlation with any metabolic enzyme and is likely adjusted to respiration-independent ROS generation. Source variations in enzyme activities are mainly observed in the anim al's gills and gut, possibly reflecting differences in the vent fluid therm al regim e and/or chemistry.