Structural Basis for the Effects of Phenylalanine on Tuning the Reduction Potential of Type 1 Copper in Azurin

A study of M13F, M44F, and G116F mutationsin azurin wasperformed to probe their effect on E & DEG;. The introductionof Phe at different locations and in varying combinations can modulate E & DEG;& PRIME; in different ways by up to 64 mV. X-raycrystal structures reveal different spatial orientations of both Pheresidues and H-bonding networks around the T1Cu-coordinating His117,which are responsible for such effects. In order to investigate the effects of the secondarycoordinationsphere in fine-tuning redox potentials (E & DEG;& PRIME;)of type 1 blue copper (T1Cu) in cupredoxins, we have introduced M13F,M44F, and G116F mutations both individually and in combination inthe secondary coordination sphere of the T1Cu center of azurin (Az)from Pseudomonas aeruginosa. Thesevariants were found to differentially influence the E & DEG;& PRIME; of T1Cu, with M13F Az decreasing E & DEG;& PRIME;, M44F Az increasing E & DEG;& PRIME;,and G116F Az showing a negligible effect. In addition, combining theM13F and M44F mutations increases E & DEG;& PRIME;by 26 mV relative to WT-Az, which is very close to the combined effectof E & DEG;& PRIME; by each mutation. Furthermore,combining G116F with either M13F or M44F mutation resulted in negativeand positive cooperative effects, respectively. Crystal structuresof M13F/M44F-Az, M13F/G116F-Az, and M44F/G116F-Az combined with thatof G116F-Az reveal these changes arise from steric effects and fine-tuningof hydrogen bond networks around the copper-binding His117 residue.The insights gained from this study would provide another step towardthe development of redox-active proteins with tunable redox propertiesfor many biological and biotechnological applications.

Automated summary

A study was conducted to investigate the effects of M13F, M44F, and G116F mutations on E & DEG; in azurin. The introduction of Phe residues at different positions and in different combinations can modulate E & DEG; in various ways, with a maximum change of 64 mV. X-ray crystal structures revealed that the spatial orientations of the Phe residues and H-bonding networks around the T1Cu-coordinating His117 are responsible for these effects. Furthermore, the study demonstrated the differential influence of M13F, M44F, and G116F mutations on the E & DEG; of T1Cu, with M13F decreasing E & DEG;, M44F increasing E & DEG;, and G116F showing a negligible effect. Combining certain mutations also resulted in cooperative effects on E & DEG;. Crystal structures provided insight into the steric effects and fine-tuning of hydrogen bond networks that contribute to these changes. Overall, the findings of this study contribute to the understanding and development of redox-active proteins with tunable properties.