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Modulation of proton-coupled electron-transfer reactions in azurin : quantum yield and pKa analyses


Long-range electron transfer (ET) reactions are central to many biochemical processes. To accomplish these long-range reactions, biomolecules often utilize multi-step ET processes via redox-active intermediates such as tryptophan and tyrosine. Azurin is a type I blue copper protein with two tyrosines (Y108 and Y72) and one tryptophan (W48), and serves as a model to study ET in proteins. Upon UV-photoexcitation of a tyrosine deficient and zinc (II)-substituted azurin mutant (ZnW48) in the presence of an exogenous electron quencher, W48 ejects an electron and forms a stable neutral radical, W48·. The quantum yield for formation of W48· depends on the identity of the exogenous quencher, namely CuIIAzurin, RuIII(NH₃)6Cl₂ , or [CoIII(NH₃)₅Cl]²⁺. In WTAzurin, the presence of the two tyrosines reduced the yield of W48· formation. This observation motivated further studies of ET reactions involving tyrosine, more specifically proton- coupled electron transfer (PCET) in which there is simultaneous transfer of the electron and phenolic proton in order to avoid high-energy intermediates. To understand potential PCET pathways, spectrophotometric titrations were performed on Y72 and Y108, both of which were found to have anomalously high pKa values. In an attempt to observe decoupled proton and electron transfer events at physiological pH values, the unnatural amino acid, nitrotyrosine, was incorporated into azurin, decreasing pKas by ~ 5 pH units. Photolysis of a sample of ZnNO₂Y72 with an exogenous quencher further decreased the yield of W48·. These studies shed light on the importance of amino acid radical intermediates in facilitating long-range electron transfer in more complex biological systems

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