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

Orange protein (ORP) is a small bacterial protein, of unknown function, that contains a unique molybdenum/copper heterometallic cluster, [S₂Mo^VIS₂Cu^IS₂Mo^VIS₂]^3- (Mo/Cu), non-covalently bound. The native cluster can be reconstituted in a protein-assisted mode by the addition of Cu^II 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 (Ala₁Ser₂His₃ 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^II in a 1:1 ratio to yield Cu^II-ORP∗, as clearly demonstrated by EPR (g_||,⊥ = 2.183, 2.042 and A^Cu_||,⊥ = 207 × 10^-4 cm^-1, 19 × 10^-4 cm^-1) and UV-visible spectroscopies (typical d-d transition bands at 520 nm, ϵ = 90 M^-1 cm^-1). The ¹H NMR spectrum shows that His₃ and His₅₃ are significantly affected upon the addition of the Cu^II. The X-ray structure shows that these two residues are very far apart (C_α-C_α ≈ 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, ¹H NMR of Ag^I-ORP∗ (Ag^I used as a surrogate of Cu^I) showed that Ag^I strongly binds to a native methionine sulfur atom rather than to the ATCUN site, suggesting that Cu^II and Cu^I have two different binding sites in ORP∗. A detailed mechanism for the formation of the Mo/Cu cluster is discussed, suggesting that Cu^II is reduced to Cu^I and transferred from the ATCUN motif to the methionine site; finally, Cu^I 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.