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Updated: Sep 8, 2025

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Published on: December 16, 2013
Tuning reduction potentials of type 1 copper center in azurin by replacing a histidine ligand with its isostructural
Yang Yu1, Nicholas M Marshall2, Dewain K Garner2
1Center of Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
Abstract:
Type 1 copper proteins have a conserved ligand set of one cysteine and two histidines, with many proteins, such as azurin, also containing an axial methionine. While the cysteine and methionine in azurin have been replaced with their respective isostructural analogues of unnatural amino acids to reveal their roles in tuning electronic structures and functional properties, such as reduction potentials (E°'), the histidine ligands have not been probed in this way. We herein report the substitution of His117 in azurin with three unnatural isostructural analogues, 5-nitrohistidine(Ntr), thiazolylalanine(SHis) and 1-methylhistidine(MeH) by expressed protein ligation. While UV-vis absorption and electron paramagnetic resonance spectroscopies confirm that isostructural replacement results in minimal structural change in the Cu(II) state, the E°' of these variants increases with increasing pKa of the δ nitrogens of the imidazole. This counter-intuitive relationship between E°' of the protein and pKa of the sidechain group suggests additional factors may play a role in tuning E°'.
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