Proton-coupled electron transfer (PCET) reactions are central to numerous biochemical processes. Azurin is a type I blue copper protein with two tyrosines (Y108 and Y72) and one tryptophan (W48), and serves as a model for PCET in proteins. Upon UV-photoexcitation, the tyrosine deficient mutant, Az48W, reduces the Cu(II) center, and forms a relatively stable neutral W48Y radical.  In wild-type azurin, the yield of W48Y is reduced significantly because of tyrosine-to-tryptophan PCET.  To understand potential PCET pathways, spectrophotometric titrations were performed on Y72 and Y108, and both tyrosines were found to have anomalously high pKa values of 11.6 and 12.5, respectively. In effort to decouple proton and electron transfer events, the unnatural amino acid, nitrotyrosine, was incorporated in azurin. The pKas were found to be 6.7 for NO2Y72 and 8.9 for NO2Y108. Radical yield measurements were also performed, and support identification of Y108 as the redox-active tyrosine residue in azurin.

Modulation of proton-coupled electron-transfer efficiencies in proteins: pKa analysis of tyrosine and nitrotyrosine in azurin

243rd National Meeting (2012)

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