This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. Background: In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT-ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim. Methods: ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy). Results: The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a K D value of 22 ± 2 nM for ZinT, while those with ZnuA point to one high affinity (K D b 20 nM) and one low affinity Zn(II) binding site (K D in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT-ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other. Conclusions: ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA. General significance: Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.

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The cyanobacterium Thermosynechococcus elongatus is one the few bacteria to possess two Dps proteins, DpsA‐Te and Dps‐Te. The present characterization of DpsA‐Te reveals unusual structural and functional features that differentiate it from Dps‐Te and the other known Dps proteins. Notably, two Zn(II) are bound at the ferroxidase center, owing to the unique substitution of a metal ligand at the A‐site (His78 in place of the canonical aspartate) and to the presence of a histidine (His164) in place of a hydrophobic residue at a metal‐coordinating distance in the B‐site. Only the latter Zn(II) is displaced by incoming iron, such that Zn(II)–Fe(III) complexes are formed upon oxidation, as indicated by absorbance and atomic emission spectroscopy data. In contrast to the typical behavior of Dps proteins, where Fe(II) oxidation by H2O2 is about 100‐fold faster than by O2, in DpsA‐Te the ferroxidation efficiency of O2 is very high and resembles that of H2O2. Oxygraphic experiments show that two Fe(II) are required to reduce O2, and that H2O2 is not released into solution at the end of the reaction. On this basis, a reaction mechanism is proposed that also takes into account the formation of Zn(II)–Fe(III) complexes. The physiological significance of the DpsA‐Te behavior is discussed in the framework of a possible localization of the protein at the thylakoid membranes, where photosynthesis takes place, with the consequent increased formation of reactive oxygen species. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7312099: DpsA (uniprotkb:http://www.uniprot.org/uniprot/Q8DL82?format=text&ascii) and DpsA (uniprotkb:http://www.uniprot.org/uniprot/Q8DL82?format=text&ascii) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by x‐ray crystallography (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114)