Beyond Blue: Systematic Modulation of Electronic Structure and Redox Properties of Type 1 Copper in Azurin

The reduction potentials of metal ions (E°′), crucial for optimizing biological processes like electron transfer and catalysis, are finely tuned by interactions between the primary and secondary coordination spheres (PCS, SCS). While previous successes in tuning E°′ in azurin have provided deeper insights into how the SCS influences electronic structure and associated redox properties of “classic” blue copper proteins, our understanding of E°′ tuning in other subclasses of type 1 Cu (T1Cu) proteins, such as green and red copper proteins, remains rudimentary. To address this issue, we report the design of a green copper center in azurin where an equatorial-to-axial shift in a histidine binding interaction leads to reorientation of the Cu-centered redox active molecular orbital and a +100 mV shift in E°′. In contrast to a 22 mV decrease in E°′ when a hydrophobic interaction is introduced in wild-type azurin through the Met13Phe mutation, this same mutation leads to a 65 mV increase in our designed green Cu azurin. More importantly, using a combination of EPR spectroscopy, protein crystallography, and quantum mechanical calculations, we uncover correlations between E°′, d–s orbital mixing, and the angle between S_Cys–Cu and N_δH46–Cu bonds, ∠(S_Cys–Cu–N_δH46), allowing rationalization of increases in E°′ of green Cu proteins through an entropically driven T-shape distortion. By providing direct connections between geometry, electronic structure, and functional properties such as E°′, this work opens previously unexplored routes to systematically modulating E°′ through the combination of spatial reorientation of the redox active molecular orbital and varying geometric distortion in the primary coordination sphere.