Modulating radical propagation in proteins by proton-coupled electron transfer and hydrogen bonding

Area of Science:

• Biochemistry and Biophysics
• Protein Electron Transfer Mechanisms
• Enzyme Catalysis

Background:

• Long-range electron transfer (ET) in proteins often involves radical relay mechanisms mediated by aromatic amino acid residues like tryptophan (Trp) and tyrosine (Tyr).
• Cytochrome c peroxidase (CcP) utilizes a W191 radical cation (W191•+) to facilitate ET from cytochrome c (Cc) to its active site.
• Modifying W191 to Tyr (Y191) reduces ET rates, but adjacent basic residues (e.g., Glu, His at E232) can restore activity by influencing radical potentials.

Purpose of the Study:

• To investigate the role of tyrosine activation by basic residues in facilitating electron transfer within CcP and its photoinitiated variant (ZnCcP).
• To elucidate the mechanisms of radical formation and propagation in CcP/ZnCcP, particularly the influence of residue 232 on Y191• formation and ET pathways.
• To explore the potential for engineering proton management strategies to control charge separation and migration in proteins.

Main Methods:

• Site-directed mutagenesis to substitute W191 with Tyr and introduce basic residues (Glu, His) at position 232.
• Spectroscopic techniques, including Electron Paramagnetic Resonance (EPR), to characterize protein radicals and their distributions.
• Photoinitiated ET studies using Zn-porphyrin CcP (ZnCcP) and kinetic measurements (pH dependence, solvent isotope effects).
• Quantum Mechanics/Molecular Mechanics (QM/MM) calculations to model radical exchange and hydrogen bonding effects.

Main Results:

• W191 substitution to Y191 significantly reduces ET rates, which are recovered by introducing adjacent basic residues (E232, H232).
• Fluorination of E232 shifts ET pH dependence, confirming that hydrogen bonding elevates the Y191• formal potential.
• In ZnCcP, proton-coupled electron transfer mechanisms facilitate Y191• formation, with distinct radical propagation patterns observed for Y191:E232 vs. Y191:H232 variants.
• EPR studies show W191•+ is at a slightly lower potential than ZnP•+ and that these radicals exchange slowly; QM/MM calculations support radical exchange and the importance of hydrogen bonds to Y191•.

Conclusions:

• Basic residues adjacent to tyrosine are crucial for activating the tyrosine radical and facilitating efficient electron transfer in CcP.
• Proton management, including hydrogen bonding and proton-coupled electron transfer, plays a critical role in modulating radical potentials and ET pathways.
• The findings have broad implications for understanding and engineering charge transfer processes in biological and potentially synthetic molecular systems.