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

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-d₅), as Trp48-d₅ in azurin, and as the photogenerated neutral tryptophan radical, Trp48-d₅^•, 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-d₅ bands relative to l-Trp-h₅ enables the analysis of vibrational markers of other amino acids, e.g., phenylalanine, that overlap with some modes of l-Trp-h₅. The Raman intensities vary between l-Trp-d₅ and l-Trp-h₅; 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-d₅^•, was generated in azurin with a slightly lower radical quantum yield than for Trp48-h₅^•. The visible resonance Raman spectrum of Trp48-d₅^• is different from that of Trp48-h₅^•, 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-d₅ is a valuable probe of vibrational structure, with minimal modification of photoreactivity and photophysics compared to l-Trp-h₅.