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

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 (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.

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

“…Transient absorption (TA) was measured using a ns-laser pump probe setup as previously described. 44 The sample was excited using a Nd/YAG laser (Quantel, Brilliant) passed through an OPO tuned to 460 nm for experiments with [Ru(dmb) 3 ] 2+ and 545 nm for experiments with [ZnTPPS] 4– . The excitation energies varied from 10 to 12 mJ/pulse for [Ru(dmb) 3 ] 2+ excitation and 22–28 mJ/pulse for [ZnTPPS] 4– excitation.…”

Computing Proton-Coupled Redox Potentials of Fluorotyrosines in a Protein Environment

“…To elucidate these local protein environments, we analyzed the hydrogen-bonding interactions of the tyrosine for each 1 μs trajectory used to compute the proton-coupled redox potentials Δ E prot‑red o (Table ). As shown in our previous work, the degree of hydrogen bonding of Y–OH to water and the protein is sensitive to the force field, and extensive sampling is required to converge the hydrogen-bonding percentages …”

“…Because oxidation of tyrosine decreases its p K a by ∼12 units, typically oxidation is accompanied by proton transfer from the phenol OH group to a nearby water molecule or protein residue via concerted PCET. Our previous MD simulations, combined with spectroscopic measurements, suggest that the proton acceptor for Y32 is a hydrogen-bonded water molecule, although a glutamate residue could not be ruled out . The small size and well-defined structure of the protein, as well as the precisely measured redox potentials, render the α 3 Y protein system ideally suited for benchmarking computational methods.…”

“…In this context, tyrosine (Y), tryptophan (W), cysteine, and/or glycine protein residues serve as high-potential one-electron redox mediators. − The mechanistic properties of Y and W are particularly interesting because these residues can efficiently transfer highly oxidizing equivalents over many Ångströms. ,,− These amino acid-based redox reactions are difficult to characterize for a number of reasons, including the very high reduction potentials ( E o ) involved, the reactive nature of the oxidized (radical) state, and the weaker optical features associated with amino acid radicals. Additionally, radical formation, transfer, and decay can be coupled to protein conformational changes occurring on a broad range of timescales. ,− For all of these reasons, detailed mechanistic information and thermodynamic information are challenging to obtain.…”

Functional and protective hole hopping in metalloenzymes

“…43,44 Oxidations of these residues also involve proton transfer processes that can modulate or limit overall reaction rates. 45 Many redox enzymes, particularly those that react with dioxygen or hydrogen peroxide generate high-potential reactive intermediates (formal potentials in the 1.0-1.25 V range) that could transfer holes to these oxidizable amino acids. Indeed, several enzymes are known to utilize hole hopping in their natural catalytic cycles.…”