Divergent Copper Radical Chemistry From an Engineered Distributed Metal Coordination Reservoir in Modular Spider Silk Proteins

Copper metalloenzymes exhibit functional diversity through precisely organized active‐site geometries. However, it remains unclear whether a disordered protein scaffold lacking a defined metal‐binding pocket can replicate this diversity. In this work, chimeric spider silk proteins RE1, RE2, and RE3 were constructed to contain one, two, and three NcMaSp2 consensus repeat units (CRU), respectively. These proteins coordinate Cu(II) ions through a distributed reservoir of histidine, tyrosine, and glutamine residues. Binding capacity scales linearly with the amount of repeat content, as shown by EPR titration and UV–Vis spectroscopy. Activation with H 2 O 2 enables three distinct radical pathways are as follows: (1) intramolecular dityrosine crosslinking (DiY fluorescence ΔF: RE3 = 17,182; RE1 = 10,016 a.u.); (2) imidazole–tyrosyl coupling, which mimics His‐Tyr cofactor biogenesis observed in cytochrome c oxidase; and (3) catechol oxidation to o‐quinone. The catechol pathway shows an inverse relationship with repeat length. The expanded CRU O‐donor network in RE3 sequesters Cu(II) and suppresses exogenous substrate oxidation. This process is restored upon the addition of 1.5 equivalents of Cu. Under crosslinking conditions, RE2 undergoes near‐quantitative insoluble aggregation, indicating a CRU content threshold where crosslinking efficiency surpasses product solubility. These results demonstrate that functional diversity in copper radical chemistry can arise from a tunable, disordered coordination reservoir rather than a specifically engineered active site. This study provides a design principle paralleling the physiological copper chemistry of the cellular prion protein.