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Abstract The key to type 1 copper (T1Cu) function lies in the fine tuning of the CuII/Ireduction potential (E°′T1Cu) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tuneE°′T1Cuin azurin over a wide range, these principles are yet to be generalized to other T1Cu‐containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) fromStreptomyces coelicolorto match the highE°′T1Cuof fungal laccases. Using ultraviolet‐visible absorption and electron paramagnetic resonance spectroscopies, together with X‐ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and correspondingE°′T1Cu. While minimal and smallE°′T1Cuincreases are observed in Y229F‐ and S292F‐SLAC, the V290N mutant exhibits a majorE°′T1Cuincrease. Moreover, the influence of these mutations onE°′T1Cuis additive, culminating in a triple mutant Y229F/V290N/S292F‐SLAC with the highestE°′T1Cuof 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (kcat/KM) relative to WT‐SLAC.
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This content will become publicly available on May 24, 2026
Contrasting secondary coordination sphere effects on spin density distribution in Red vs. Blue Cu azurin
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.
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- Award ID(s):
- 2420683
- PAR ID:
- 10596688
- Publisher / Repository:
- Springer Nature
- Date Published:
- Journal Name:
- JBIC Journal of Biological Inorganic Chemistry
- ISSN:
- 1432-1327
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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