Two Tryptophans Are Better Than One in Accelerating Electron Flow through a Protein

Area of Science:

• Biophysical Chemistry
• Protein Engineering
• Electron Transfer Mechanisms

Background:

• Azurin is a small blue copper protein involved in electron transfer.
• Rhenium (Re) photosensitizers can be used to initiate electron transfer in proteins.
• Understanding long-range electron transfer is crucial for bioenergetics and enzyme function.

Purpose of the Study:

• To investigate the effect of inserting two tryptophan residues on electron transfer dynamics in a Pseudomonas aeruginosa azurin mutant.
• To characterize the mechanism and efficiency of photoinitiated electron transfer from a Re photosensitizer to the copper center.
• To explore the role of multistep tunneling in protein-based electron transfer.

Main Methods:

• Construction and structural characterization of a Pseudomonas aeruginosa azurin mutant (Re126WWCu) with two tryptophan residues.
• Time-resolved spectroscopy, including luminescence, visible, and infrared absorption, to monitor electron transfer events.
• Quantum mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) simulations to analyze electron transfer pathways and solvation dynamics.

Main Results:

• Photoexcited Re label initiated Cu(I) oxidation in Re126WWCu with a ~70 ns time constant.
• Time-resolved spectroscopy revealed two rapid reversible electron transfer steps: W124 to *Re and W122 to W124•+.
• A rate-determining Cu(I) oxidation by W122•+ occurred ~11 Å away, followed by recombination within 120 μs.

Conclusions:

• Multistep tunneling through the double-tryptophan chain confers a significant advantage (~9000-fold) over single-step tunneling.
• The findings support the hypothesis that hole-hopping via tryptophan/tyrosine chains protects enzymes from oxidative damage.
• Protein structure and dynamics play a critical role in modulating long-range electron transfer efficiency.