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Metalloproteins tune the electronic properties of metal active sites through a combination of primary and secondary coordination sphere effects (PCS and SCS) to efficiently perform an array of redox chemistry, including electron transfer (ET) and catalysis. A major influence of these effects is the anisotropic spatial distribution of redox-active molecular orbitals (RAMOs), which in turn dictates redox chemistry of the metalloproteins. While much progress has been made in understanding the SCS effects on RAMOs in individual native metalloproteins, it has been difficult to experimentally examine the influence of the same SCS effects on RAMOs with different spatial distributions. Taking advantage of our recent studies of SCS effect on blue copper azurin from Pseudomonas aeruginosa (Blue CuAz) and its M121H/H46E variant that closely mimic the red copper protein (Red CuAz), in which their RAMOs are dominated by either Cu-S or Cu-S interactions, respectively, we herein compare and contrast how the same SCS modifications impact the electronic and geometric structures of blue and red Cu center in the same protein scaffold. Specifically, we expand our understanding of unpaired electron distribution at the Cu-binding site of Red CuAz and its SCS N47S, F114P, and F114N mutations using 1H and 14N electron-nuclear double resonance (ENDOR) spectroscopy, and then further combine these data sets with recent studies and DFT calculations to provide insight into how these mutations differentially (or similarly) impact electronic structure in Red vs. Blue CuAz. We find that electrostatics produce similar effects in both Red and Blue CuAz, where the introduction of dipole moments in the vicinity of Cu and S produce changes in spin density distribution and of the same sign and comparable magnitude. However, disruption of H-bonding with S through the F114P mutation leads to opposing effects in Red vs. Blue CuAz, which we propose arise from differences in the conformation of Cys112 sidechain adapted in the absence the stabilizing SC112•••H-N backbone interaction. 

Type 1 copper (T1Cu) centers are crucial in biological electron transfer (ET) processes, exhibiting a wide range of reduction potentials (E°′T1Cu) to match their redox partners and optimize ET rates. While tuning E°′T1Cu in mononuclear T1Cu proteins like azurin has been successful, it is more difficult for multicopper oxidases. Specifically, while replacing axial methionine to leucine in azurin increased its E°′T1Cu by ~100 mV, the corresponding M298L mutation in small laccase from Streptomyces coelicolor (SLAC) unexpectedly decreased its E°′T1Cu by 12 mV. X-ray crystallography revealed two axial water molecules in M298L-SLAC, leading to the decrease of E°′T1Cu due to decreased hydrophobicity. Structural alignment and molecular dynamics simulation indicated a key difference in T1Cu axial loop position, leading to the different outcome upon methionine to leucine mutation. Based on structural analyses, we introduced additional F195L and I200F mutations, leading to partial removal of axial waters, a 122-mV increase in E°′T1Cu, and a 7-fold increase in kcat/KM from M298L-SLAC. These findings highlight the complexity of tuning E°′T1Cu in multicopper oxidases and provide valuable insights into how structure-based protein engineering can contribute to the broader understanding of T1Cu center, E°′T1Cu and reactivity tuning for applications in solar energy transfer, fuel cells, and bioremediation.