Proton-Coupled Electron Transfer from Tyrosine in
the Interior of a de novo Protein: Mechanisms and
Primary Proton Acceptor
Posted on 2020-06-17 - 21:29
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
(kPCET) 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. kPCET 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.
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Nilsen-Moe, Astrid; Reinhardt, Clorice R.; Glover, Starla D.; Liang, Li; Hammes-Schiffer, Sharon; Hammarström, Leif; et al. (2020). Proton-Coupled Electron Transfer from Tyrosine in
the Interior of a de novo Protein: Mechanisms and
Primary Proton Acceptor. ACS Publications. Collection. https://doi.org/10.1021/jacs.0c04655