Intramolecular electron transfer in single-site-mutated azurins

Area of Science:

• Biochemistry
• Protein Electron Transfer
• Spectroscopy

Background:

• Azurin is a blue, single-copper protein from Pseudomonas aeruginosa.
• Electron transfer in proteins is fundamental to biological processes.
• Understanding mutation effects on protein dynamics is key to protein engineering.

Purpose of the Study:

• To investigate the impact of single-site mutations on electron transfer in azurin.
• To elucidate the role of specific residues in intramolecular electron transfer pathways.
• To determine reorganization energy and electron transfer driving force.

Main Methods:

• Pulse radiolysis experiments to study electron transfer kinetics.
• Site-directed mutagenesis to create specific azurin variants.
• Temperature dependence studies to analyze reaction rates.

Main Results:

• The disulfide group in azurin mutants was reduced by CO2- radicals at rates similar to the native protein.
• Reoxidation of the RSSR- radical showed intramolecular electron transfer rates between 30-70 s-1.
• Mutation of Phe114 to Ala nearly doubled the electron transfer rate, suggesting increased driving force.
• Mutations at Trp48 showed minimal effect on electron transfer rates.
• Pathway calculations indicated a longer, through-backbone path is more efficient.

Conclusions:

• Phe114 plays a significant role in modulating electron transfer rates in azurin, likely by influencing the driving force.
• Trp48 appears to be marginally involved in the primary electron transfer pathway.
• The study provides insights into protein electron transfer mechanisms and the impact of structural modifications.