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

The reversible Y–O^•/Y–OH redox properties of the α₃Y 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 α₃Y, by <i>in vivo</i> 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 p<i>K</i> (8.0 ± 0.1) of the unnatural residue. Square-wave voltammetry on α₃(3,5)­F₂Y provides an <i>E</i>°′(Y–O^•/Y–OH) of 1026 ± 4 mV versus the normal hydrogen electrode (pH 5.70 ± 0.02) and shows that the fluoro substitutions lower the <i>E</i>°′ by −30 ± 3 mV. These results illustrate the utility of combining the optimized α₃Y tyrosine radical system with <i>in vivo</i> 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 <i>E°′</i> 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 <i>Escherichia coli</i> ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues

Pourbaix Diagram, Proton-Coupled Electron Transfer, and Decay Kinetics of a Protein Tryptophan Radical: Comparing the Redox Properties of W₃₂^• and Y₃₂^• Generated Inside the Structurally Characterized α₃W and α₃Y Proteins

Protein-based “hole” hopping typically involves spatially arranged redox-active tryptophan or tyrosine residues. Thermodynamic information is scarce for this type of process. The well-structured α₃W model protein was studied by protein film square wave voltammetry and transient absorption spectroscopy to obtain a comprehensive thermodynamic and kinetic description of a buried tryptophan residue. A Pourbaix diagram, correlating thermodynamic potentials (<i><i>E</i>°</i>′) with pH, is reported for W₃₂ in α₃W and compared to equivalent data recently presented for Y₃₂ in α₃Y (Ravichandran, K. R.; Zong, A. B.; Taguchi, A. T.; Nocera, D. G.; Stubbe, J.; Tommos, C. J. Am. Chem. Soc. 2017, 139, 2994−3004). The α₃W Pourbaix diagram displays a p<i>K</i>_OX of 3.4, a <i><i>E</i>°</i>′(W₃₂(N^•+/NH)) of 1293 mV, and a <i><i>E</i>°</i>′(W₃₂(N^•/NH); pH 7.0) of 1095 ± 4 mV versus the normal hydrogen electrode. W₃₂(N^•/NH) is 109 ± 4 mV more oxidizing than Y₃₂(O^•/OH) at pH 5.4–10. In the voltammetry measurements, W₃₂ oxidation–reduction occurs on a time scale of about 4 ms and is coupled to the release and subsequent uptake of one full proton to and from bulk. Kinetic analysis further shows that W₃₂ oxidation likely involves pre-equilibrium electron transfer followed by proton transfer to a water or small water cluster as the primary acceptor. A well-resolved absorption spectrum of W₃₂^• is presented, and analysis of decay kinetics show that W₃₂^• persists ∼10⁴ times longer than aqueous W^• due to significant stabilization by the protein. The redox characteristics of W₃₂ and Y₃₂ are discussed relative to global and local protein properties

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

<i>Escherichia coli</i> class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y₁₂₂•) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y₁₂₂ ⇆ [W₄₈] ⇆ Y₃₅₆ in β2 to Y₇₃₁ ⇆ Y₇₃₀ ⇆ C₄₃₉ in α2). Equilibration of Y₃₅₆•, Y₇₃₁•, and Y₇₃₀• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y₁₂₂• and Y₃₅₆• has not been detected. Our recent report of Y₃₅₆• formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y₁₂₂ (F₃Y₁₂₂•-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F₃Y₁₂₂• and Y₃₅₆•. Incubation of F₃Y₁₂₂•-β2, Y₇₃₁F-α2 (or Y₇₃₀F-α2), CDP, and ATP at different temperatures (2–37 °C) provides Δ<i>E</i>°′(F₃Y₁₂₂•–Y₃₅₆•) of 20 ± 10 mV at 25 °C. The pH dependence of the F₃Y₁₂₂• ⇆ Y₃₅₆• interconversion (pH 6.8–8.0) reveals that the proton from Y₃₅₆ is in rapid exchange with solvent, in contrast to the proton from Y₁₂₂. Insertion of 3,5-difluorotyrosine (F₂Y) at Y₃₅₆ and rapid freeze-quench EPR analysis of its reaction with Y₇₃₁F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F₂Y₃₅₆• generation by the native Y₁₂₂•. F_<i>n</i>Y-RNRs (<i>n</i> = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y₁₂₂ and C₄₃₉ is ∼200 meV uphill

Photochemical Tyrosine Oxidation in the Structurally Well-Defined α₃Y Protein: Proton-Coupled Electron Transfer and a Long-Lived Tyrosine Radical

Tyrosine oxidation–reduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The α₃Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of α₃Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in α₃Y. Time-resolved spectroscopy using a soluble flash-quench generated [Ru­(2,2′-bipyridine)₃]³⁺ oxidant provides high-quality Y32–O• absorption spectra. The rate constant of Y32 oxidation (<i>k</i>_PCET) is pH dependent: 1.4 × 10⁴ M^–1 s^–1 (pH 5.5), 1.8 × 10⁵ M^–1 s^–1 (pH 8.5), 5.4 × 10³ M^–1 s^–1 (pD 5.5), and 4.0 × 10⁴ M^–1 s^–1 (pD 8.5). <i>k</i>^H/<i>k</i>^D of Y32 oxidation is 2.5 ± 0.5 and 4.5 ± 0.9 at pH­(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32–O• is 28–58% versus the concentration of [Ru­(2,2′-bipyridine)₃]³⁺. Y32–O• decays slowly, <i>t</i>_1/2 in the range of 2–10 s, at both pH 5.5 and 8.5, via radical–radical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32–O• is discussed relative to the structural properties of the Y32 site. Finally, the static α₃Y NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32–O• come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of α₃Y

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

Redox-active tyrosines (Ys) play essential roles in enzymes involved in primary metabolism including energy transduction and deoxynucleotide production catalyzed by ribonucleotide reductases (RNRs). Thermodynamic characterization of Ys in solution and in proteins remains a challenge due to the high reduction potentials involved and the reactive nature of the radical state. The structurally characterized α₃Y model protein has allowed the first determination of formal reduction potentials (<i>E</i>°′) for a Y residing within a protein (Berry, B. W.; Martı́nez-Rivera, M. C.; Tommos, C. <i>Proc. Natl. Acad. Sci. U. S. A.</i> <b>2012</b>, <i>109</i>, 9739–9743). Using Schultz’s technology, a series of fluorotyrosines (F_<i>n</i>Y, <i>n</i> = 2 or 3) was site-specifically incorporated into α₃Y. The global protein properties of the resulting α₃(3,5)­F₂Y, α₃(2,3,5)­F₃Y, α₃(2,3)­F₂Y and α₃(2,3,6)­F₃Y variants are essentially identical to those of α₃Y. A protein film square-wave voltammetry approach was developed to successfully obtain reversible voltammograms and <i>E</i>°’s of the very high-potential α₃F_<i>n</i>Y proteins. <i>E</i>°′(pH 5.5; α₃F_<i>n</i>Y­(O•/OH)) spans a range of 1040 ± 3 mV to 1200 ± 3 mV versus the normal hydrogen electrode. This is comparable to the potentials of the most oxidizing redox cofactors in nature. The F_<i>n</i>Y analogues, and the ability to site-specifically incorporate them into any protein of interest, provide new tools for mechanistic studies on redox-active Ys in proteins and on functional and aberrant hole-transfer reactions in metallo-enzymes. The former application is illustrated here by using the determined α₃F_<i>n</i>Y Δ<i>E</i>°’s to model the thermodynamics of radical-transfer reactions in F_<i>n</i>Y-RNRs and to experimentally test and support the key prediction made

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

3-Aminotyrosine (NH₂Y) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NH₂Y of key importance for its application in mechanistic studies. By combining the tRNA/NH₂Y-RS suppression technology with a model protein tailored for amino acid redox studies (α₃X, X = NH₂Y), the formal reduction potential of NH₂Y₃₂(O^•/OH) (<i><i>E</i>°′</i> = 395 ± 7 mV at pH 7.08 ± 0.05) could be determined using protein film voltammetry. We find that the Δ<i><i>E</i>°′</i> between NH₂Y₃₂(O^•/OH) and Y₃₂(O^•/OH) when measured under reversible conditions is ∼300–400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NH₂Y and Y. We have also generated D₆-NH₂Y₇₃₁-α2 of ribonucleotide reductase (RNR), which when incubated with β2/CDP/ATP generates the D₆-NH₂Y₇₃₁^•-α2/β2 complex. By multifrequency electron paramagnetic resonance (35, 94, and 263 GHz) and 34 GHz ¹H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establish RNH₂^• planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NH₂Y-RNR incorporated by infidelity of the tRNA/NH₂Y-RS pair was determined by a generally useful LC-MS method. This information is essential to the utility of this NH₂Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH₂Y-substituted RNRs