Proton-Coupled Electron Transfer from Tyrosine in the Interior of a de novo Protein: Mechanisms and Primary Proton Acceptor

Proton-coupled electron transfer (PCET) from tyrosine produces a neutral tyrosyl radical (Y^•) that is vital to many catalytic redox reactions. To better understand how the protein environment influences the PCET properties of tyrosine, we have studied the radical formation behavior of Y₃₂ in the α₃Y model protein. The previously solved α₃Y solution NMR structure shows that Y₃₂ is sequestered ∼7.7 ± 0.3 Å below the protein surface without any primary proton acceptors nearby. Here we present transient absorption kinetic data and molecular dynamics (MD) simulations to resolve the PCET mechanism associated with Y₃₂ oxidation. Y₃₂^• was generated in a bimolecular reaction with [Ru­(bpy)₃]³⁺ formed by flash photolysis. At pH > 8, the rate constant of Y₃₂^• formation (k_PCET) increases by one order of magnitude per pH unit, corresponding to a proton-first mechanism via tyrosinate (PTET). At lower pH < 7.5, the pH dependence is weak and shows a previously measured KIE ≈ 2.5, which best fits a concerted mechanism. k_PCET is independent of phosphate buffer concentration at pH 6.5. This provides clear evidence that phosphate buffer is not the primary proton acceptor. MD simulations show that one to two water molecules can enter the hydrophobic cavity of α₃Y and hydrogen bond to Y₃₂, as well as the possibility of hydrogen-bonding interactions between Y₃₂ and E₁₃, through structural fluctuations that reorient surrounding side chains. Our results illustrate how protein conformational motions can influence the redox reactivity of a tyrosine residue and how PCET mechanisms can be tuned by changing the pH even when the PCET occurs within the interior of a protein.