Protein-Assisted Formation of Molybdenum Heterometallic Clusters: Evidence for the Formation of S₂MoS₂-M-S₂MoS₂ Clusters with M = Fe, Co, Ni, Cu, or Cd within the Orange Protein

The Orange Protein (ORP) is a small bacterial protein, of unknown function, that harbors a unique molybdenum/copper (Mo/Cu) heterometallic cluster, [S₂Mo^VIS₂Cu^IS₂Mo^VIS₂]^3-, noncovalently bound. The apo-ORP is able to promote the formation and stabilization of this cluster, using Cu^II- and Mo^VIS₄ ^2- salts as starting metallic reagents, to yield a Mo/Cu-ORP that is virtually identical to the native ORP. In this work, we explored the ORP capability of promoting protein-assisted synthesis to prepare novel protein derivatives harboring molybdenum heterometallic clusters containing iron, cobalt, nickel, or cadmium in place of the “central” copper (Mo/Fe-ORP, Mo/Co-ORP, Mo/Ni-ORP, or Mo/Cd-ORP). For that, the previously described protein-assisted synthesis protocol was extended to other metals and the Mo/M-ORP derivatives (M = Cu, Fe, Co, Ni, or Cd) were spectroscopically (UV-visible and electron paramagnetic resonance (EPR)) characterized. The Mo/Cu-ORP and Mo/Cd-ORP derivatives are stable under oxic conditions, while the Mo/Fe-ORP, Mo/Co-ORP, and Mo/Ni-ORP derivatives are dioxygen-sensitive and stable only under anoxic conditions. The metal and protein quantification shows the formation of 2Mo:1M:1ORP derivatives, and the visible spectra suggest that the expected {S₂MoS₂MS₂MoS₂} complexes are formed. The Mo/Cu-ORP, Mo/Co-ORP, and Mo/Cd-ORP are EPR-silent. The Mo/Fe-ORP derivative shows an EPR S = ³/₂ signal (E/D ≈ 0.27, g ≈ 5.3, 2.5, and 1.7 for the lower M= ±¹/₂ doublet, and g ≈ 5.7 and 1.7 (1.3 predicted) for the upper M = ±³/₂ doublet), consistent with the presence of either one S = ⁵/₂ Fe^III antiferromagnetically coupled to two S = ¹/₂ Mo^V or one S = ³/₂ Fe^I and two S = 0 Mo^VI ions, in both cases in a tetrahedral geometry. The Mo/Ni-ORP shows an EPR axial S = ¹/₂ signal consistent with either one S = ¹/₂ Ni^I and two S = 0 Mo^VI or one S = ¹/₂ Ni^III antiferromagnetically coupled to two S = ¹/₂ Mo^V ions, in both cases in a square-planar geometry. The Mo/Cu-ORP and Mo/Cd-ORP are described as {Mo^VI-Cu^I-Mo^VI} and {Mo^VI-Cd^II-Mo^VI}, respectively, while the other derivatives are suggested to exist in at least two possible electronic structures, {Mo^VI-M^I-Mo^VI} ↔ {Mo^V-M^III-Mo^V}.