Abstract
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.
Highlights
Protein redox chemistry is at the heart of many biologically important processes such as photosynthesis, respiration, and nitrogen fixation
The large oxidoreductase class of enzymes uses a range of organic molecules and metallocofactors for catalytic and long-range electron transfer (ET) reactions.[1−7] Some oxidoreductases use tyrosine (Y), tryptophan (W), cysteine, glycine, and possibly methionine as high potential one-electron redox mediators.[7−18] Y and W are of particular interest because these residues can form spatially organized chains in which high potential, one-electron oxidizing equivalents are moved over large distances.[14−18] Gray and Winkler have even suggested that Y/W-based radical transfer (“hole hopping”) pathways may be quite common and potentially serve as an important protective mechanism against oxidative damage.[17,18]
ET from Y is typically coupled to proton transfer (PT) in a protoncoupled electron transfer (PCET) reaction, resulting in the formation of a neutral radical species.[8,10,19−23] The thermodynamics and kinetics involved in radical formation and decay in these amino acids have direct implications for biocatalytic multistep ET/PCET processes
Summary
Protein redox chemistry is at the heart of many biologically important processes such as photosynthesis, respiration, and nitrogen fixation. Due to the complexity and size of many enzymes, it is extremely challenging to experimentally resolve the thermodynamic and kinetic behavior of a single amino-acid residue. The α3X family of model proteins bridges the gap between small-molecule model systems and enzymes by providing a well-defined protein environment wherein the formation of a single amino-acid radical can be experimentally resolved.[44] the α3X proteins provide a unique
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