期刊
BIOCHEMISTRY
卷 52, 期 49, 页码 8907-8915出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi401494f
关键词
-
资金
- National Institutes of Health [GM079190, GM29595]
The reversible Y-O-center dot/Y-OH redox properties of the alpha Y-3 model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in alpha Y-3, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH values below the apparent pK (8.0 +/- 0.1) of the unnatural residue. Square-wave voltammetry on alpha(3)(3,5)F2Y provides an E degrees'(Y-O-center dot/Y-OH) of 1026 +/- 4 mV versus the normal hydrogen electrode (pH 5.70 +/- 0.02) and shows that the fluoro substitutions lower the E degrees' by -30 +/- 3 mV. These results illustrate the utility of combining the optimized alpha Y-3 tyrosine radical system with in vivo nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein E degrees' values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is notable because solution potentials have been the main thermodynamic data available for amino acid radicals. The findings in this paper are discussed relative to recent mechanistic studies of the multistep radical-transfer process in Escherichia coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues.
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