Unexpected effect of an axial ligand mutation in the type 1 copper center in small laccase: structure-based analyses and engineering to increase reduction potential and activity

Abstract

Type 1 copper (T1Cu) centers are crucial in biological electron transfer (ET) processes, exhibiting a wide range of reduction potentials to match their redox partners and optimize ET rates. While tuning 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 by ∼100 mV, the corresponding M298L mutation in small laccase from Streptomyces coelicolor (SLAC) unexpectedly decreased its by 12 mV. X-ray crystallography revealed two axial water molecules in M298L-SLAC, leading to the decrease of 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 , and a 7-fold increase in k _cat/K _M from M298L-SLAC. These findings highlight the complexity of tuning in multicopper oxidases and provide valuable insights into how structure-based protein engineering can contribute to the broader understanding of T1Cu center, and reactivity tuning for applications, such as in solar energy transfer, fuel cells, and bioremediation.

Fig. 1. (A) Electronic absorption spectrum and (B) EPR spectrum (black: experimental spectra; red: fitted spectra) of SLAC mutants involved in this study. Data for WT-SLAC is replotted from ref. . LF-SLAC stands for F195L/I200F-SLAC.

Fig. 2. (A) for mutants involved in this paper. All were measured in 50 mM MOPS, 150 mM NaCl, pH 7 buffer. ^†Data from ref. . (B) Relative change between each mutant.

Fig. 3. (A) Crystal structure of M298L-SLAC (PDB: 9NJI) showing the axial water (red spheres), with a 2F_O – F_C electron density contoured at 1 RMSD; (B) Structure alignment between M298L-SLAC (purple, PDB: 9NJI) and N47S/M121L-azurin (yellow, PDB: 3JT2 (ref. 8)). The red arrow indicates the shift of the loop that holds the axial leucine. The red spheres indicate the axial water in M298L-SLAC.

Fig. 4. Two conformations (I and II) that the axial leucine can adopt from MD simulation of. (A) M298L-SLAC; (B) N47S/M121L-Az.

Fig. 5. Crystal structure of LF-M298L-SLAC (PDB: 9NJJ) showing the two F195L conformations in subfigure (A) and (B). The axial water is shown in red spheres.

Fig. 6. (A) Michaelis–Menten parameters for mutants involved in this paper using ABTS as substrate. ^†Data from ref. . (Michaelis–Menten parameters were measured in 50 mM MOPS, 150 mM NaCl, pH 7 at 310 K.) Histogram demonstrating (B) k_cat, and (C) k_cat/K_M for different mutants.