Formal Reduction Potential of 3,5-Difluorotyrosine in a Structured Protein: Insight into Multistep Radical Transfer

Abstract

The reversible Y-O•/Y-OH redox properties of the α3Y model protein enable 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 α3Y, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH below the apparent pK (8.0 ± 0.1) of the unnatural residue. Square-wave voltammetry on α3(3,5)F2Y provides an E°′(Y-O•/Y-OH) of 1026 ± 4 mV versus the NHE (pH 5.70 ± 0.02) and shows that the fluoro-substitutions lower E°′ by –30 ± 3 mV. These results illustrate the utility of combining the optimized α3Y 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 significant since solution potentials have been the main thermodynamic data available for amino-acid radicals. These findings are discussed relative to recent mechanistic studies on the multistep radical-transfer process in E. coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues.

A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes

“…Rapid freeze-quench (RFQ)-EPR spectroscopy of the reaction between F 2 Y 356 -β2, Y 731 F-α2, CDP, and ATP at pH 8.2 and 25 °C revealed F 2 Y 356 • at 3 ± 1% of the total radical concentration. This observation provided a Δ E °′(F 2 Y 356 •–Y 122 •) of 70 ± 5 mV, which along with our recent measurement of the reduction potential of F 2 Y in a protein environment 23,25 gives an estimate of Δ E °′(Y 356 •–Y 122 •) of ∼100 mV at pH 7.6. The results of the site specifically incorporated unnatural amino acids described herein together with our previous studies allow us to propose a thermodynamic landscape for the RT pathway in the E. coli class Ia RNR that is ∼200 meV uphill between Y 122 and C 439 .…”

Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm

“…30 Voltammetry characterization of the 2e − α 3 NH 2 Y redox system differs relative to the previously studied 1e − α 3 Y, 16, 30 2-mercaptophenol-α 3 C, 51 the α 3 F n Ys, 15, 16 and α 3 W 52 proteins. The combination of a less oxidizing potential range and a 2e − current, yielded protein film cyclic voltammograms of excellent quality (e.g.…”

“…37 The α 3 scaffold increases the radical t 1/2 dramatically (×10 4 for Y 32 • and W 32 • relative to Y• (aq) and W• (aq) 42, 52 and has allowed reversible voltammograms and E °’ values to be obtained on Y 32 , W 32 and a number of tyrosine analogs. 15, 16, 30, 51, 52 …”

“…4–7 In the latter case, fluorotyrosines (F n Y, n = 2, 3) 8–11 and 3-aminotyrosine (NH 2 Y) 12, 13 have served as particularly useful mechanistic probes. F n Ys display a broad range of p K a ’s (6.4–7.8) 10, 14–16 and formal reduction potentials E °’ (−25 to +135 mV) relative to Y, 15, 16 which are important to assessing proton coupled electron transfer (PCET) mechanisms. Reduction potentials reported by DeFelippis et al in 1991 17 suggested that NH 2 Y could serve as a −190 to −290 mV “radical trap” 12, 13 and provide evidence for Y• intermediates that are normally difficult to detect because of their high reactivity and short half-lives ( t 1/2 ).…”

Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer

“…44,45 SWV was used to obtain formal E °′(radical/amino acid) protein potentials for α 3 Y, 33 2MP-α 3 C, 28 and α 3 (3,5)F 2 Y. 30 The α 3 X potentials are pH dependent and measurements performed at low pH are more difficult due to the higher potentials and more pronounced background currents involved. 33 SWV on α 3 (2,3,5)F 3 Y, α 3 (2,3)F 2 Y, and α 3 (2,3,6)F 3 Y was predicted to be challenging since measurements had to be done ~ pH 5.5 (≥ 1.7 pH units below the p K app ’s of the α 3 F n Y proteins) and test studies on α 3 (2,3,5)F 3 Y placed E °′ well above +1 V. Voltammetry data from an on-going parallel project involving an α 3 (NH 2 )Y variant (Lee, Nocera, Stubbe and Tommos) suggested that a protein film approach, where the protein is introduced to the surface of the working electrode outside the electrochemical cell, improves the Faradaic response of the α 3 X system.…”