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

Abstract

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 (kPCET) 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). kH/kD 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, t1/2 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.

Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY

“…A very similar lifetime of TyrO ⅐ has been achieved in ␣3Y, a de novo model protein specifically designed for investigating the stabilizing effect of the protein matrix on the lifetime of tyrosyl radicals (38). ␣3Y does not contain any cofactors and places the reactive tyrosine in a three helix bundle scaffold.…”

“…Similar to aCRY, ␣Y3 does not carry a covalent modification of tyrosine or a metal center for stabilization of TyrO ⅐ (39). However, the residue is buried deeply in a hydrophobic environment in ␣3Y without any hydrogen bonding partner or access to the solvent (38). Therefore, the surrounding protein matrix is rather different compared with the one present in aCRY, considering the relatively exposed position of Tyr-373 in a cavity on the surface of the protein, as derived from the modeled structure (16).…”

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

“…40–42, 51, 52 These proteins share a common three-helix bundle (α 3 ) with a redox site at position 32 (X 32 ) in the protein interior. 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 …”

Proton-Coupled Electron Transfer in Tyrosine and a β-Hairpin Maquette: Reaction Dynamics on the Picosecond Time Scale

“…24,25 Ruthenium photosensitizers were also used to study the kinetics of tyrosyl radical formation and decay in the model peptide α3, which forms an α-helical bundle. 26 A β-hairpin tyrosine-containing model has also been described; it is an 18-mer peptide that has been designed de novo and is inspired by PSII. 27,28 Peptide A is an 18-mer and contains a single tyrosine (Y5) and cross-strand histidine (H14).…”