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Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping.

ABSTRACT: Transient tyrosine and tryptophan radicals play key roles in the electron transfer (ET) reactions of photosystem (PS) II, ribonucleotide reductase (RNR), photolyase, and many other proteins. However, Tyr and Trp are not functionally interchangeable, and the factors controlling their reactivity are often unclear. Cytochrome c peroxidase (CcP) employs a Trp191•+ radical to oxidize reduced cytochrome c (Cc). Although a Tyr191 replacement also forms a stable radical, it does not support rapid ET from Cc. Here we probe the redox properties of CcP Y191 by non-natural amino acid substitution, altering the ET driving force and manipulating the protic environment of Y191. Higher potential fluorotyrosine residues increase ET rates marginally, but only addition of a hydrogen bond donor to Tyr191• (via Leu232His or Glu) substantially alters activity by increasing the ET rate by nearly 30-fold. ESR and ESEEM spectroscopies, crystallography, and pH-dependent ET kinetics provide strong evidence for hydrogen bond formation to Y191• by His232/Glu232. Rate measurements and rapid freeze quench ESR spectroscopy further reveal differences in radical propagation and Cc oxidation that support an increased Y191• formal potential of ?200 mV in the presence of E232. Hence, Y191 inactivity results from a potential drop owing to Y191•+ deprotonation. Incorporation of a well-positioned base to accept and donate back a hydrogen bond upshifts the Tyr• potential into a range where it can effectively oxidize Cc. These findings have implications for the YZ/YD radicals of PS II, hole-hopping in RNR and cryptochrome, and engineering proteins for long-range ET reactions.

SUBMITTER: Yee EF

PROVIDER: S-EPMC7043243 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping.

Yee Estella F EF Dzikovski Boris B Crane Brian R BR

Journal of the American Chemical Society 20191028 44

Transient tyrosine and tryptophan radicals play key roles in the electron transfer (ET) reactions of photosystem (PS) II, ribonucleotide reductase (RNR), photolyase, and many other proteins. However, Tyr and Trp are not functionally interchangeable, and the factors controlling their reactivity are often unclear. Cytochrome c peroxidase (CcP) employs a Trp191•+ radical to oxidize reduced cytochrome c (Cc). Although a Tyr191 replacement also forms a stable radical, it does ...[more]

PMID: 31603693

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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.

Dataset Information

Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping.

Publications

Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping.

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