Insights into the Molybdenum/Copper Heterometallic Cluster Assembly in the Orange Protein: Probing Intermolecular Interactions with an Artificial Metal-Binding ATCUN Tag.

Inorg Chem

UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa , 2829-516 Caparica, Portugal.

Published: August 2017

Orange protein (ORP) is a small bacterial protein, of unknown function, that contains a unique molybdenum/copper heterometallic cluster, [SMoSCuSMoS] (Mo/Cu), non-covalently bound. The native cluster can be reconstituted in a protein-assisted mode by the addition of Cu plus tetrathiomolybdate to apo-ORP under controlled conditions. In the work described herein, we artificially inserted the ATCUN ("amino terminus Cu and Ni") motif in the Desulfovibrio gigas ORP (AlaSerHis followed by the native amino acid residues; modified protein abbreviated as ORP*) to increase our understanding of the Mo/Cu cluster assembly in ORP. The apo-ORP* binds Cu in a 1:1 ratio to yield Cu-ORP*, as clearly demonstrated by EPR (g = 2.183, 2.042 and A = 207 × 10 cm, 19 × 10 cm) and UV-visible spectroscopies (typical d-d transition bands at 520 nm, ε = 90 M cm). The H NMR spectrum shows that His and His are significantly affected upon the addition of the Cu. The X-ray structure shows that these two residues are very far apart (C-C ≈ 27.9 Å), leading us to suggest that the metal-induced NMR perturbations are due to the interaction of two protein molecules with a single metal ion. Docking analysis supports the metal-mediated dimer formation. The subsequent tetrathiomolybdate binding, to yield the native Mo/Cu cluster, occurs only upon addition of dithiothreitol, as shown by UV-visible and NMR spectroscopies. Additionally, H NMR of Ag-ORP* (Ag used as a surrogate of Cu) showed that Ag strongly binds to a native methionine sulfur atom rather than to the ATCUN site, suggesting that Cu and Cu have two different binding sites in ORP*. A detailed mechanism for the formation of the Mo/Cu cluster is discussed, suggesting that Cu is reduced to Cu and transferred from the ATCUN motif to the methionine site; finally, Cu is transferred to the cluster-binding region, upon the interaction of two protein molecules. This result may suggest that copper trafficking is triggered by redox-dependent coordination properties of copper in a trafficking pathway.

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http://dx.doi.org/10.1021/acs.inorgchem.7b00840DOI Listing

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