Raman and Quantum Yield Studies of Trp48-d₅ in Azurin: Closed-Shell and Neutral Radical Species

Abstract

Isotopologues are valuable vibrational probes that shift features in a vibrational spectrum while preserving the electronic structure of the molecule. We report the vibrational and electronic spectra of perdeuterated tryptophan in solution (l-Trp-d 5), as Trp48-d 5 in azurin, and as the photogenerated neutral tryptophan radical, Trp48-d 5 •, in azurin. The UV resonance Raman bands of the perdeuterated closed-shell tryptophan in solution and in azurin are lower in frequency relative to the protiated counterpart. The observed decrease in frequencies of l-Trp-d 5 bands relative to l-Trp-h 5 enables the analysis of vibrational markers of other amino acids, e.g., phenylalanine, that overlap with some modes of l-Trp-h 5. The Raman intensities vary between l-Trp-d 5 and l-Trp-h 5; these differences likely reflect modifications in normal mode composition upon perdeuteration. Analysis of the W3, W6, and W17 modes suggests that the W3 mode retains its utility as a conformational marker; however, the H-bond markers W6 and W17 appear to be less sensitive upon perdeuteration. The neutral tryptophan radical, Trp48-d 5 •, was generated in azurin with a slightly lower radical quantum yield than for Trp48-h 5 •. The visible resonance Raman spectrum of Trp48-d 5 • is different from that of Trp48-h 5 •, especially in terms of relative intensities, and all assignable peaks decreased in frequency upon perdeuteration. The absorption and emission spectra of the perdeuterated closed-shell and radical species exhibited hypsochromic shifts of less than 1 nm relative to the protiated species. The data presented here indicate that l-Trp-d 5 is a valuable probe of vibrational structure, with minimal modification of photoreactivity and photophysics compared to l-Trp-h 5.

Role of the Triplet State and Protein Dynamics in the Formation and Stability of the Tryptophan Radical in an Apoazurin Mutant

“…The kinetics from Figure 5 were analyzed with Scheme I in which the triplet state is the precursor to the neutral radical. Figure 7 shows the kinetics of radical formation in terms of fractional population of radical; despite the previous observation that the radical quantum yield for ZnAzW48 depends on the incident power, with decreased yields at high powers, 58 all six powers were included in the fit. The poor fit at high power is apparent.…”

“…Several studies have characterized the tryptophan neutral radical in a blue-copper protein, azurin, of Pseudomonas aeruginosa . − Azurin is a 128-residue protein with eight β-strands arranged in a Greek key motif and a small α-helical segment. The metal-binding site is located at one end of the protein, with a single native tryptophan residue (W48) buried in a hydrophobic pocket.…”

“…Studies on W48 revealed that the tryptophan radical can be photogenerated via a covalently bound photoactive label or direct UV-excitation of the Zn­(II)-substituted protein (Zn-azurin). − The mechanism for formation is expected to involve photoejection of an electron from closed-shell tryptophan to form the cation radical, followed by proton transfer to generate the neutral radical. The resulting neutral radical in a tyrosine-deficient Zn-azurin is a long-lived species that is stable for several hours in an oxygen-free environment .…”

Plasmonically Generated Tryptophan Radical Anion on Gold Nanoparticles Investigated by Combined Surface-Enhanced Raman Scattering and Density Functional Theory Calculations

“…The presence of Trp •– was unexpected. The Trp radical cation (Trp •+ ) is commonly reported in the protein literature, where Trp serves as an electron donor. ,− Indeed, others have used NATA alongside Trp to study the radical cation due to their similarities in the structure and the ability to associate changes with the indole ring moiety. The similarities in the SERS spectrum of Trp and NATA confirm that the signal obtained results from the indole ring structure.…”

Photoinduced Electron Transfer from the Tryptophan Triplet State in Zn-Azurin

“…In the former case, the ligand-to-metal charge transfer (LMCT) band at 628 nm provides a spectroscopic signature that reflects the efficiency of the ET step separate from the proton transfer (PT) reaction. 21 Furthermore, the long lifetime of W48• (7.3 h) and the characteristic absorption peak around 515 nm make it possible to quantify the radical quantum yield with steady state absorbance measurements. 19 The mechanistic details of W48• formation in Zn-azurin are not fully understood but may involve photoinduced PCET between the triplet excited state, 3 W48*, and an external electron acceptor.…”