Formal reduction potential of 3,5-difluorotyrosine in a structured protein: insight into multistep radical transfer.
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abstract
The reversible Y-O/Y-OH redox properties of the 3Y 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 3Y, 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 (8.00.1) of the unnatural residue. Square-wave voltammetry on 3(3,5)F2Y provides an E'(Y-O/Y-OH) of 10264 mV versus the normal hydrogen electrode (pH 5.700.02) and shows that the fluoro substitutions lower the E' by -303 mV. These results illustrate the utility of combining the optimized 3Y 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' 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.
