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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 ?3Y model protein. The previously solved ?3Y 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 ?3Y 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.

SUBMITTER: Nilsen-Moe A

PROVIDER: S-EPMC7315633 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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

Nilsen-Moe Astrid A Reinhardt Clorice R CR Glover Starla D SD Liang Li L Hammes-Schiffer Sharon S Hammarström Leif L Tommos Cecilia C

Journal of the American Chemical Society 20200617 26

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 ...[more]

PMID: 32479070

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Project description: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.

Project description:The coupling between electrons and protons and the long-range transport of protons play important roles throughout biology. Biomimetic systems derived from benzimidazole-phenol (BIP) constructs have been designed to undergo proton-coupled electron transfer (PCET) upon electrochemical or photochemical oxidation. Moreover, these systems can transport protons along hydrogen-bonded networks or proton wires through multiproton PCET. Herein, the nonequilibrium dynamics of both single and double proton transfer in BIP molecules initiated by oxidation are investigated with first-principles molecular dynamics simulations. Although these processes are concerted in that no thermodynamically stable intermediate is observed, the simulations predict that they are predominantly asynchronous on the ultrafast time scale. For both systems, the first proton transfer typically occurs ∼100 fs after electron transfer. For the double proton transfer system, typically the second proton transfer occurs hundreds of femtoseconds after the initial proton transfer. A machine learning algorithm was used to identify the key molecular vibrational modes essential for proton transfer: a slow, in-plane bending mode that dominates the overall inner-sphere reorganization, the proton donor-acceptor motion that leads to vibrational coherence, and the faster donor-hydrogen stretching mode. The asynchronous double proton transfer mechanism can be understood in terms of a significant mode corresponding to the two anticorrelated proton donor-acceptor motions, typically decreasing only one donor-acceptor distance at a time. Although these PCET processes appear concerted on the time scale of typical electrochemical experiments, attaching these BIP constructs to photosensitizers may enable the detection of the asynchronicity of the electron and multiple proton transfers with ultrafast two-dimensional spectroscopy. Understanding the fundamental PCET mechanisms at this level will guide the design of PCET systems for catalysis and energy conversion processes.

Dataset Information

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

Publications

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

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