Reduction potential tuning at a type 1 copper site does not compromise electron transfer reactivity

Area of Science:

• Biochemistry
• Bioinorganic Chemistry
• Protein Science

Background:

• Type 1 (T1) copper sites are vital for biological electron transfer (ET).
• These sites feature conserved His2Cys equatorial ligands and variable axial ligands, including methionine (Met), glutamine (Gln), or absence of a ligand.
• Cucumber basic protein (CBP) is a plantacyanin (PLT) with a T1 copper site.

Purpose of the Study:

• To investigate the impact of axial ligand variation on the electron self-exchange (ESE) rate constant and reduction potential of T1 copper sites.
• To understand how modulating the axial coordination environment affects the intrinsic ET reactivity of proteins like CBP.

Main Methods:

• Site-directed mutagenesis was used to create Met89Gln and Met89Val variants of CBP.
• Electron self-exchange (ESE) rate constants (k(ese)) were measured to assess intrinsic ET reactivity.
• Reduction potentials were measured to evaluate changes in the electronic properties of the copper site.

Main Results:

• The Met89Gln variant of CBP exhibited an approximately 7-fold decrease in k(ese) compared to wild-type.
• The Met89Val mutation resulted in a 2-fold increase in k(ese).
• Decreasing axial interaction strength (Gln > Met > Val) enhanced ESE reactivity by approximately one order of magnitude and increased reduction potential by approximately 350 mV.

Conclusions:

• The axial coordination environment of T1 copper sites significantly influences ET reactivity and reduction potential.
• Variable axial coordination provides a mechanism for tuning the driving force of ET to optimize interactions with biological partners.
• Enhanced reactivity in three-coordinate T1 copper sites, as seen in fungal laccases and Fet3p, likely facilitates intramolecular ET.