The blue copper protein, azurin, is an electron transfer protein involved in the denitrification processes in some bacteria. The single tryptophan residue in the 48th position (W48) has been shown to undergo a proton coupled electron transfer (PCET) reaction in a stepwise manner, where a transient cation radical is generated after the loss of an electron, followed by proton transfer (PT) to form a neutral radical. The W48 residue in azurin is buried in a hydrophobic pocket of the protein, with no exposure to solvent; hence, there is no obvious PT path. The crystal structure indicates that the H+ of the indole ring of W48 is near a phenylalanine residue (F110), possibly in a cation- π like interaction. We hypothesize that after photoexcitation and subsequent formation of the cation radical, the proton is transferred to F110 via interaction with the π cloud, and is then further transferred to T17, a residue only ~5Å away from F110. To investigate this pathway, we studied two azurin mutants, T17V and T17I, to determine if the presence of a hydrophobic residue, like valine or isoleucine, hindered PT from the cation radical, leading to decreased formation of W48 neutral radical. Via experiments, we were able to demonstrate that the mutants have a lowered ratio of PT compared to the control, suggesting that hydrophobic residues hinder the transfer of proton from the cation radical released to the solvent via the hypothesized pathway.

Mutagenic study of the mechanism of proton transfer during the proton coupled electron transfer in Azurin

ACS Spring 2022

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