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

ZITARE, ULISES A.; ESPINOZA-CARA, ANDRÉS; SZUSTER, JONATHAN; VILA, ALEJANDRO J.; SANTALLA, MARÍA C.; MURGIDA, DANIEL H.

Groningen

Simposio; 14th International Symposium on Biocatalysis and Biotransformations (BioTrans 2019); 2019

Copper sites in proteins have evolved to perform either electron transfer or redox catalysis. Type 1 (T1) and Cu sites are electron transfer hubs bound to a A rigid protein fold that prevents binding of exogenous ligands and side reactions. Here I report the engineering of several T1 sites by loop-directed mutagenesis within a Cu scaffold with unique electronic structures and functional features. A Also, these sites display high reduction potentials and the ability to bind exogenous ligands such as imidazole and azide, forming a Type 2 like adduct. UV-Vis, Resonant Raman, EPR and electrochemical (EQ) experiments allowed the study of such structural, electronic and redox properties, as well as the thermodynamic and kinetic characterization of adduct formation for one case.