Tyrosine and tryptophan play critical roles in facilitating proton-coupled electron transfer (PCET) processes essential to life. The local protein environment is anticipated to modulate the thermodynamics of amino acid radicals to achieve controlled, unidirectional PCET. Herein, square-wave voltammetry was employed to investigate the electrostatic effects on the redox properties of tryptophan in two variants of the protein azurin. Each variant contains a single redox-active tryptophan, W48 or W108, in a unique and buried protein environment. These tryptophan residues exhibit reversible square-wave voltammograms. A Pourbaix plot, representing the reduction potentials versus pH, is presented for the non-H-bonded W48, which has potentials comparable to those of tryptophan in solution. The reduction potentials of W108 are seen to be increased by more than 100 mV across the same pH range. Molecular dynamics shows that, despite its buried indole ring, the N–H of W108 hydrogen bonds with a water cluster, while W48 is completely excluded from interactions with water or polar groups. These redox properties provide insight into the role of the protein in tuning the reactivity of tryptophan radicals, a requirement for controlled biological PCET.

Fluorinated 5-hydroxytryptophans (Fn-5HOWs) were synthesized in gram scale quantities and incorporated into β-hairpin peptides and the protein azurin. The redox-active Fn-5HOWs exhibit unique radical spectroscopic signatures that expand the function...

Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials (E°) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 mode�considered a vibrational marker for hydrogen bonding�and E°is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/ frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases E°and reveal properties of the protein microenvironment surrounding tryptophan.

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