DNA-binding proteins from starved cells (Dps, EC: 1.16.3.1) have a variety of different biochemical activities such as DNA-binding, iron sequestration, and HO detoxification. Most bacteria commonly feature one or two Dps enzymes, whereas the cyanobacterium displays an unusually high number of five Dps proteins (NpDps1-5). Our previous studies have indicated physiological differences, as well as cell-specific expression, among these five proteins. Three of the five NpDps proteins, NpDps1, -2, and -3, were classified as canonical Dps proteins. To further investigate their properties and possible importance for physiological function, here we characterized and compared them Nondenaturing PAGE, gel filtration, and dynamic light-scattering experiments disclosed that the three NpDps proteins exist as multimeric protein species in the bacterial cell. We also demonstrate Dps-mediated iron oxidation catalysis in the presence of HO However, no iron oxidation with O as the electron acceptor was detected under our experimental conditions. In modeled structures of NpDps1, -2, and -3, protein channels were identified that could serve as the entrance for ferrous iron into the dodecameric structures. Furthermore, we could demonstrate pH-dependent DNA-binding properties for NpDps2 and -3. This study adds critical insights into the functions and stabilities of the three canonical Dps proteins from and suggests that each of the Dps proteins within this bacterium has a specific biochemical property and function.

Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H 2 O 2 . At their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe 2+ to reduce H 2 O 2 and therefore play an essential role in the protection against oxidative stress and maintaining iron homeostasis. Whereas most bacteria accommodate one or two Dps, there are five different Dps proteins in Nostoc punctiforme , a phototrophic and filamentous cyanobacterium. This uncommonly high number of Dps proteins implies a sophisticated machinery for maintaining complex iron homeostasis and for protection against oxidative stress. Functional analyses and structural information on cyanobacterial Dps proteins are rare, but essential for understanding the function of each of the NpDps proteins. In this study, we present the crystal structure of NpDps4 in its metal-free, iron- and zinc-bound forms. The FOC coordinates either two iron atoms or one zinc atom. Spectroscopic analyses revealed that NpDps4 could oxidize Fe 2+ utilizing O 2 , but no evidence for its use of the oxidant H 2 O 2 could be found. We identified Zn 2+ to be an effective inhibitor of the O 2 -mediated Fe 2+ oxidation in NpDps4. NpDps4 exhibits a FOC that is very different from canonical Dps, but structurally similar to the atypical one from DpsA of Thermosynechococcus elongatus . Sequence comparisons among Dps protein homologs to NpDps4 within the cyanobacterial phylum led us to classify a novel FOC class: the His-type FOC. The features of this special FOC have not been identified in Dps proteins from other bacterial phyla and it might be unique to cyanobacterial Dps proteins.

Highlights  The dodecamer stability of the two atypical NpDps are pH dependent.  Both NpDps4 and NpDps5 show DNA binding properties.  Both NpDps4 and NpDps5 show binding properties upon interaction with NpFdx proteins.  NpDps5, with structural similarities to bacterioferritins, is also related to Dps proteins, due to its dodecameric structure and DNA binding ability.

25Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H 2 O 2 . At 26 their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe 2+ to reduce H 2 O 2 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 36 Fe 2+ utilizing O 2 , but no evidence for its use of the oxidant H 2 O 2 could be found. We identified 37 Zn 2+ to be an effective inhibitor of the O 2 -mediated Fe 2+ 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 3 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 (Fe 2+ ) to ferric (Fe 3+ ) 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.58To reach the catalytic center, the Fe 2+ 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. 61The 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, i...

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