Expression and Spectroscopic Characterization of Deuterated Tryptophan in Azurin

Electron transfer (ET) is an important reaction in biological processes such as photosynthesis and respiration. Previous studies have shown that aromatic amino acids, including tryptophan, can serve as hopping sites for long-range ET. The blue copper protein, azurin, is a model protein for the study of tryptophan in a biological ET reaction. Azurin contains a single tryptophan residue (W48) in a hydrophobic pocket that undergoes a photo-induced proton coupled electron transfer (PCET) reaction to generate a long-lived neutral radical. The motivation for this work is to investigate isotopologues in the PCET reaction. A minimal media expression protocol was optimized to produce azurin with perdeuterated tryptophan (W48-d5) in higher yields than previous effort. This was achieved by inclusion of an extended overnight expression at room temperature. Additional isotopologues in which the NH group of the indole ring was exchanged to ND were also generated by following an established protocol. Spectroscopic characterization through UV resonance Raman spectroscopy confirmed that L-Trp-d5 and ND-L-trp-d5 were successfully incorporated as NH-W48-d5, ND-W48-h5, and ND-W48-d5 in azurin. These variants had similar fluorescence quantum yields (within 10%). However, the phosphorescence quantum yields varied, with the W48-d5 variants exhibiting lower yields than W48-h5. Azurin with NH-W48-d5 also displayed decreased quantum yield for ET and the formation of the neutral radical compared to NH-W48-h5. Preliminary data on the ND variants (ND-W48-h5, and ND-W48-d5) were inconclusive because of the inability to reproduce results from the control sample; future experiments will be needed