Switching the proton-coupled electron transfer mechanism for non-canonical tyrosine residues in a de novo protein.

The proton-coupled electron transfer (PCET) reactions of tyrosine (Y) are instrumental to many redox reactions in nature. This study investigates how the local environment and the thermodynamic properties of Y influence its PCET characteristics. Herein, 2- and 4-mercaptophenol (MP) are placed in the well-folded α3C protein (forming 2MP-α3C and 4MP-α3C) and oxidized by external light-generated [Ru(L)3]3+ complexes. The resulting neutral radicals are long-lived (>100 s) with distinct optical and EPR spectra. Calculated spin-density distributions are similar to canonical Y˙ and display very little spin on the S-S bridge that ligates the MPs to C32 inside the protein. With 2MP-α3C and 4MP-α3C we probe how proton transfer (PT) affects the PCET rate constants and mechanisms by varying the degree of solvent exposure or the potential to form an internal hydrogen bond. Solution NMR ensemble structures confirmed our intended design by displaying a major difference in the phenol OH solvent accessible surface area (≤∼2% for 2MP and 30-40% for 4MP). Additionally, 2MP-C32 is within hydrogen bonding distance to a nearby glutamate (average O-O distance is 3.2 ± 0.5 Å), which is suggested also by quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations. Neither increased exposure of the phenol OH to solvent (buffered water), nor the internal hydrogen bond, was found to significantly affect the PCET rates. However, the lower phenol pKa values associated with the MP-α3C proteins compared to α3Y provided a sufficient change in PT driving force to alter the PCET mechanism. The PCET mechanism for 2MP-α3C and 4MP-α3C with moderately strong oxidants was predominantly step-wise PTET for pH values, but changed to concerted PCET at neutral pH values and below when a stronger oxidant was used, as found previously for α3Y. This shows how the balance of ET and PT driving forces is critical for controlling PCET mechanisms. The presented results improve our general understanding of amino-acid based PCET in enzymes.

Concerted and Stepwise Proton-Coupled Electron Transfer for Tryptophan-Derivative Oxidation with Water as the Primary Proton Acceptor: Clarifying a Controversy.

Concerted electron-proton transfer (CEPT) reactions avoid charged intermediates and may be energetically favorable for redox and radical-transfer reactions in natural and synthetic systems. Tryptophan (W) often partakes in radical-transfer chains in nature but has been proposed to only undergo sequential electron transfer followed by proton transfer when water is the primary proton acceptor. Nevertheless, our group has shown that oxidation of freely solvated tyrosine and W often exhibit weakly pH-dependent proton-coupled electron transfer (PCET) rate constants with moderate kinetic isotope effects (KIE ≈ 2-5), which could be associated with a CEPT mechanism. These results and conclusions have been questioned. Here, we present PCET rate constants for W derivatives with oxidized Ru- and Zn-porphyrin photosensitizers, extracted from laser flash-quench studies. Alternative quenching/photo-oxidation methods were used to avoid complications of previous studies, and both the amine and carboxylic acid groups of W were protected to make the indole the only deprotonable group. With a suitably tuned oxidant strength, we found an ET-limited reaction at pH < 4 and weakly pH-dependent rates at pH > ∼5 that are intrinsic to the PCET of the indole group with water (H2O) as the proton acceptor. The observed rate constants are up to more than 100 times higher than those measured for initial electron transfer, excluding the electron-first mechanism. Instead, the reaction can be attributed to CEPT. These conclusions are important for our view of CEPT in water and of PCET-mediated radical reactions with solvent-exposed tryptophan in natural systems.

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 (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.