Some N(2)-fixing bacteria prolong the functionality of nitrogenase in molybdenum starvation by a special Mo storage protein (MoSto) that can store more than 100 Mo atoms. The presented 1.6 Å X-ray structure of MoSto from Azotobacter vinelandii reveals various discrete polyoxomolybdate clusters, three covalently and three noncovalently bound Mo(8), three Mo(5-7), and one Mo(3) clusters, and several low occupied, so far undefinable clusters, which are embedded in specific pockets inside a locked cage-shaped (αβ)(3) protein complex. The structurally identical Mo(8) clusters (three layers of two, four, and two MoO(n) octahedra) are distinguishable from the [Mo(8)O(26)](4-) cluster formed in acidic solutions by two displaced MoO(n) octahedra implicating three kinetically labile terminal ligands. Stabilization in the covalent Mo(8) cluster is achieved by Mo bonding to Hisα156-N(ε2) and Gluα129-O(ε1). The absence of covalent protein interactions in the noncovalent Mo(8) cluster is compensated by a more extended hydrogen-bond network involving three pronounced histidines. One displaced MoO(n) octahedron might serve as nucleation site for an inhomogeneous Mo(5-7) cluster largely surrounded by bulk solvent. In the Mo(3) cluster located on the 3-fold axis, the three accurately positioned His140-N(ε2) atoms of the α subunits coordinate to the Mo atoms. The formed polyoxomolybdate clusters of MoSto, not detectable in bulk solvent, are the result of an interplay between self- and protein-driven assembly processes that unite inorganic supramolecular and protein chemistry in a host-guest system. Template, nucleation/protection, and catalyst functions of the polypeptide as well as perspectives for designing new clusters are discussed.

A continuous FeMo cofactor supply for nitrogenase maturation is ensured in Azotobacter vinelandii by developing a cage‐like molybdenum storage protein (MoSto) capable to store ca. 120 molybdate molecules (MoO42−) as discrete polyoxometalate (POM) clusters. To gain mechanistic insight into this process, MoSto was characterized by Mo and ATP/ADP content, structural, and kinetic analysis. We defined three functionally relevant states specified by the presence of both ATP/ADP and POM clusters (MoStofunct), of only ATP/ADP (MoStobasal) and of neither ATP/ADP nor POM clusters (MoStozero), respectively. POM clusters are only produced when ATP is hydrolyzed to ADP and phosphate. Vmax was ca. 13 μmolphosphate·min−1·mg−1 and Km for molybdate and ATP/Mg2+ in the low micromolar range. ATP hydrolysis presumably proceeds at subunit α, inferred from a highly occupied α‐ATP/Mg2+ and a weaker occupied β‐ATP/no Mg2+‐binding site found in the MoStofunct structure. Several findings indicate that POM cluster storage is separated into a rapid ATP hydrolysis‐dependent molybdate transport across the protein cage wall and a slow molybdate assembly induced by combined auto‐catalytic and protein‐driven processes. The cage interior, the location of the POM cluster depot, is locked in all three states and thus not rapidly accessible for molybdate from the outside. Based on Vmax, the entire Mo storage process should be completed in less than 10 s but requires, according to the molybdate content analysis, ca. 15 min. Long‐time incubation of MoStobasal with nonphysiological high molybdate amounts implicates an equilibrium in and outside the cage and POM cluster self‐formation without ATP hydrolysis. Databases The crystal structures MoSto in the MoSto‐F6, MoSto‐F7, MoStobasal, MoStozero, and MoSto‐F1vitro states were deposited to PDB under the accession numbers PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GU5, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GUJ, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GWB, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GWV, and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6GX4.

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Pyoverdines (PVDs) are important chromophore-containing siderophores of fluorescent pseudomonad bacteria such as the opportunistic human pathogen in which they play an essential role in host infection. PVD biosynthesis encompasses a complex pathway comprising cytosolic nonribosomal peptide synthetases that produce a polypeptide precursor that periplasmic enzymes convert to the final product. The structures of most enzymes involved in PVD chromophore maturation have been elucidated, but the structure of the essential tyrosinase PvdP, a monooxygenase required for the penultimate step in PVD biosynthesis, is not known. Here, we closed this gap by determining the crystal structure of PvdP in an apo and tyrosine-complexed state at 2.1 and 2.7 Å, respectively. These structures revealed that PvdP is a homodimer, with each chain consisting of a C-terminal tyrosinase domain and an N-terminal eight-stranded β-barrel reminiscent of streptavidin that appears to have a structural role only. We observed that ligand binding leads to the displacement of a "placeholder" tyrosine that blocks the active site in the apo structure. This exposes a large, deep binding site that seems suitable for accommodating ferribactin, a substrate of PvdP in PVD biosynthesis. The binding site consists almost exclusively of residues from the tyrosinase domain. Of note, we also found that this domain is more closely related to tyrosinases from arthropods rather than to tyrosinases from other bacteria. In conclusion, our work unravels the structural basis of PvdP's activity in PVD biosynthesis, observations that may inform structure-guided development of PvdP-specific inhibitors to manage infections.

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