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The Dps4 from Nostoc Punctiforme ATCC 29133 Is a Member of His-Type FOC Containing Dps

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The Dps4 from Nostoc Punctiforme ATCC 29133 Is a Member of His-Type FOC Containing Dps bioRxiv preprint doi: https://doi.org/10.1101/656892; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 The Dps4 from Nostoc punctiforme ATCC 29133 is a member of His-type FOC containing Dps 3 protein class that can be broadly found among cyanobacteria 4 5 Christoph Howe1, Vamsi K. Moparthi1#, Felix M. Ho1, Karina Persson2* and Karin Stensjö1* 6 7 1Department of Chemistry-Ångström Laboratory, Uppsala University, Uppsala, Sweden 8 2Department of Chemistry, Umeå University, Umeå, Sweden 9 10 #Current address: Department of Physics, Chemistry and Biology, Division of Chemistry, 11 Linköping University, Linköping, Sweden. 12 13 *Corresponding authors: 14 E-mail: [email protected] (KS), [email protected] (KP) 15 16 17 18 19 20 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/656892; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 Abstract 25 Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H2O2. At 2+ 26 their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe to reduce H2O2 27 and therefore play an essential role in the protection against oxidative stress and maintaining 28 iron homeostasis. Whereas most bacteria accommodate one or two Dps, there are five 29 different Dps proteins in Nostoc punctiforme, a phototrophic and filamentous cyanobacterium. 30 This uncommonly high number of Dps proteins implies a sophisticated machinery for 31 maintaining complex iron homeostasis and for protection against oxidative stress. Functional 32 analyses and structural information on cyanobacterial Dps proteins are rare, but essential for 33 understanding the function of each of the NpDps proteins. In this study, we present the crystal 34 structure of NpDps4 in its metal-free, iron- and zinc-bound forms. The FOC coordinates either 35 two iron atoms or one zinc atom. Spectroscopic analyses revealed that NpDps4 could oxidize 2+ 36 Fe utilizing O2, but no evidence for its use of the oxidant H2O2 could be found. We identified 2+ 2+ 37 Zn to be an effective inhibitor of the O2-mediated Fe oxidation in NpDps4. NpDps4 exhibits 38 a FOC that is very different from canonical Dps, but structurally similar to the atypical one from 39 DpsA of Thermosynechococcus elongatus. Sequence comparisons among Dps protein 40 homologs to NpDps4 within the cyanobacterial phylum led us to classify a novel FOC class: 41 the His-type FOC. The features of this special FOC have not been identified in Dps proteins 42 from other bacterial phyla and it might be unique to cyanobacterial Dps proteins. 43 Keywords: ferroxidase center, iron, oxidative stress, crystal structure, reactive oxygen 44 species 45 46 Introduction 47 Dps proteins (DNA-binding proteins from starved cells), also referred to as miniferritins, are 48 only found in prokaryotes and belong to the class of ferritin-like proteins [1], alongside with 2 bioRxiv preprint doi: https://doi.org/10.1101/656892; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 49 bacterioferritins (bfr) and ferritins (ftn). Dps proteins exhibit a remarkable three-dimensional 50 structure consisting of twelve monomers (or six dimers), forming a spherically shaped protein 51 complex with a hollow spherical interior [2,3]. 52 On the inside, each dimeric interface creates two identical catalytic centers, called the 53 ferroxidase centers (FOC). There, the oxidation of ferrous iron (Fe2+) to ferric (Fe3+) takes 54 place and an iron oxide mineral core consisting of up to 500 Fe atoms can be formed [4]. 55 Canonical FOCs in Dps proteins consist of five conserved amino acids, namely two His and 56 one Asp from one monomer as well as one Glu and one Asp from the adjacent monomer at 57 the dimer interface. 58 To reach the catalytic center, the Fe2+ ions have been suggested to travel through two types 59 of pores that are connecting the internal cavity with the exterior [2]. One pore type is the ferritin- 60 like pore, which is named after their structural similarity to the iron entrance pores in ferritins. 61 The ferritin-like pore has been frequently assigned to be the iron entrance pore due to its 62 negatively charged character in canonical Dps structures. The other pore type, the Dps-type 63 pore, is specific to Dps proteins. Compared to the ferritin-like pore it has a more hydrophobic 64 character and its function remains unknown [5]. 2+ 65 When Fe has entered the Dps and bound to the FOC, it is usually oxidized by O2 or H2O2. 66 However in canonical Dps proteins, O2 is utilized at a 100-fold lower reaction rate as compared 67 to H2O2, as demonstrated for Escherichia coli Dps (EcDps) [4,6]. Due to the consumption of 2+ 68 H2O2 and Fe a mineral core is formed and the Fenton reaction is circumvented, from which 69 in the absence of the Dps protein highly toxic hydroxyl radicals are produced [7]. 70 Thus the Dps protein class joins the cellular protection machinery alongside catalases and 71 peroxidases that disarm reactive oxygen species (ROS) [8,9]. Cyanobacteria, which contain 72 the O2-evolving photosynthetic apparatus, require an effective response to ROS, as O2 is the 73 precursor for all ROS including H2O2 [10,11]. The filamentous diazotrophic cyanobacterium 74 Nostoc punctiforme ATTC 29133 exhibits a complex H2O2-protection system including three 3 bioRxiv preprint doi: https://doi.org/10.1101/656892; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 75 catalases and various peroxidases [12]. In addition to those, we have identified five Dps 76 proteins [13]. 77 In cyanobacteria the existence of multiple Dps proteins has been found to be rather common 78 [13,14]. But most of the more well-studied Dps proteins originate from pathogens that possess 79 only one or two Dps proteins [2,15–17]. Information about cyanobacterial Dps proteins and 80 their involvement in iron homeostasis and ROS defense is scarce. To learn specifically more 81 about cyanobacterial Dps proteins, we have collected data on the different NpDps from N. 82 punctiforme in the past years to identify their physiological roles. In an earlier study [13] we 83 hypothesized that, despite all belonging to the Dps protein family, they might differ in their 84 biochemical properties and thus in their physiological function. 85 First we have identified that, with the exception of Npdps2, all the Npdps genes are 86 predominantly transcribed in heterocysts [18], a specific cell type that is developed in a regular 87 pattern along the filament during nitrogen depletion. In heterocysts a microoxic environment 88 is created, necessitated by the oxygen sensitivity of the nitrogenase that is involved in N2- 89 fixation [19]. In an earlier in vivo study we additionally observed that NpDps2 (Npun_F3730) 90 is an essential protectant against H2O2 that is exogenously added to the cyanobacterial culture 91 [13]. Interestingly, biochemical investigations showed that NpDps1, NpDps2 and NpDps3 2+ 3+ 92 could catalyze the oxidation reaction of Fe to Fe with H2O2, but not with O2 [20]. NpDps1- 93 3 showed high sequence similarities with the canonical Dps [20]. In silico experiments 94 indicated that NpDps1-3 likely import Fe2+ through their ferritin-like pores as commonly 95 hypothesized for typical Dps proteins [20]. Additionally we discovered that NpDps2 and 96 NpDps5 (Npun_F6212), are involved in the tolerance against high light intensities [21], a 97 condition that enhances the production of ROS [22,23]. 98 The information about NpDps4 is limited and its role in N. punctiforme is not resolved [13]. 99 NpDps4 shares a sequence identity of ca. 64 % (based on the sequences derived by 100 Cyanobase [24] and alignment by Clustal Omega [25]) with the DpsA from the cyanobacterium 4 bioRxiv preprint doi: https://doi.org/10.1101/656892; this version posted May 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 101 Thermosynechoccus elongatus (TeDpsA). Notably, T. elongatus exhibits two Dps proteins, 102 which are the only cyanobacterial Dps proteins that have been structurally studied so far. 103 While TeDps was found to share high similarities with canonical Dps [5], TeDpsA was found 104 to be an atypical Dps. TeDpsA has been reported to exhibit surprisingly similar Fe2+ oxidation 2+ 105 rates with H2O2 and O2, which was suggested to relate to Zn bound at the FOC [14]. 106 Canonical FOCs are usually dominated by negatively charged carboxyl groups from various 107 Glu and Asp among two conserved His [14]. Instead, the FOC of TeDpsA was found to have 108 a non-canonical nature with two additional His and less involvement of carboxyl groups from 109 Asp or Glu.
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