Engineering a bifunctional copper site in the cupredoxin fold by loop-directed mutagenesis

Abstract

Loop directed mutagenesis leads to a cupredoxin withthe strongest copper–thiolate bond known to date, high reduction potential and imidazole binding properties.

CuA-based chimeric T1 copper sites allow for independent modulation of reorganization energy and reduction potential

“…assigned to S Met / Cu 2+ and pseudo-s S Cys / Cu 2+ LMCT transitions. 36 The 3 450 /3 600 ratios of the chimeras range from 1.10 to 1.64 (Table S1 and Fig. S1 †), thus approaching the value reported for nitrite reductase, 1.74, 42 which is the most perturbed T1 site reported to date.…”

“…1) following established procedures. 36 The sequences were chosen aiming to cover a wide range of geometric distortions, from classic axial blue sites, such as azurin and amicyanin, to strongly perturbed rhombic green sites, such as pseudoazurin, cucumber basic protein and nitrite reductase. Thus, the ligand loops of the chimeras differ in length and sequence, but contain a preserved Cys/His/Met T1 ligand set, which is completed with His75 from the Tt-Cu A scaffold.…”

“…In contrast, the absorption spectra of all the chimeras display two partially overlapping strong bands at around 400-450 nm that are 38 on the left, and that of Ami-Tt-Cu A on the right (PDB ID 5U7N). 36 The loop replaced in the chimeras is indicated in green and orange, respectively. The loop sequences employed to obtain the different chimeras are listed below.…”

“…[24][25][26][27][28][29][30] This methodology has also allowed for the successful insertion of Cu A sites into the scaffold of T1 [31][32][33][34][35] proteins and vice versa. 36 So far, most efforts have focused on tuning reduction potentials (E 0 ) of T1-like mononuclear centers through rst and second sphere perturbations, attaining up to 700 mV modulation. 1,15 The tuning of kinetic ET parameters such as the reorganization energy (l) received signicantly less attention for metalloproteins in general and for T1 sites in particular, 21,37 and no clear patterns have been established for the simultaneous or independent modulation of l and E 0 .…”

“… 24 – 30 This methodology has also allowed for the successful insertion of Cu A sites into the scaffold of T1 31 – 35 proteins and vice versa . 36 …”