An outer-sphere hydrogen-bond network constrains copper coordination in blue proteins

Area of Science:

• Biochemistry
• Bioinorganic Chemistry
• Protein Engineering

Background:

• Blue copper proteins, like azurins, exhibit low reduction potentials due to protein structure.
• Hydrogen bonds stabilize copper ligands, influencing protein reduction potentials.
• Protein framework constraints tune copper reduction potentials away from typical complexes.

Purpose of the Study:

• To investigate the impact of altering hydrogen bond networks on amicyanin's reduction potential.
• To explore how relaxing constraints on copper(I) geometry affects protein function.
• To engineer amicyanin variants with modified electrochemical properties.

Main Methods:

• Site-directed mutagenesis of the amicyanin ligand loop in *P. denitrificans*.
• Electrochemical analysis to determine reduction potentials (E(0)) of wild-type and mutant proteins.
• Spectroscopic methods to assess changes in ligand coordination and histidine pK(a).

Main Results:

• Mutations significantly increased the reduction potential of amicyanin variants (e.g., to 415 mV) compared to the wild type (265 mV).
• Altered hydrogen bonding networks relaxed constraints on copper(I) ligation geometry.
• Mutations shifted the pK(a) of a key histidine residue from 7.0 to below 5.

Conclusions:

• Protein structure, particularly hydrogen bond networks, plays a crucial role in tuning blue copper protein reduction potentials.
• Modifying the ligand environment provides a strategy to engineer the electrochemical properties of metalloproteins.
• These findings offer insights into the bioenergetics of electron transfer in copper-containing proteins.