Structural Examination of the Transient 3-Aminotyrosyl Radical on the PCET Pathway of E. coli Ribonucleotide Reductase by Multifrequency EPR Spectroscopy

Abstract

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y122•) within the β2 subunit to a cysteine residue (C439) within the α2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y122 → W48 → Y356 in β2, across the subunit interface to Y731 → Y730 → C439 in α2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in α2. Incubation of this mutant with β2, substrate, and allosteric effector resulted in loss of the Y122• and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH2Y•). In the current study [15N]- and [14N]-NH2Y730• have been generated in H2O and D2O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH2Y730•. The g tensor (gx = 2.0052, gy = 2.0042, gz = 2.0022), the orientation of the β-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH2 moiety are consistent with an intramolecular hydrogen bond within NH2Y730•. This analysis is an essential first step in using the detailed structure of NH2Y730• to formulate a model for a PCET mechanism within α2 and for use of NH2Y in other systems where transient Y•s participate in catalysis.

Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm

“…25–27 These results are described in Figures S8 and S9 and demonstrate that the trigonal pyramidal conformation can potentially increase the reduction potential by 190 – 390 mV.…”

“…48, 49 The P2 ε 280 nm was predicted from the peptide sequence where the ε 280 nm of 1900 M −1 cm −1 for NH 2 Y was previously determined in H 2 O/HCl, pH 1. 25 The peptide standard curve was constructed by analyzing 0.1 ng to 10 ng of each peptide by ESI-MS.…”

“…Incubation of each NH 2 Y-α2 (or NH 2 Y-β2) mutant with the appropriate second subunit, substrate, and effector resulted in formation of an aminotyrosyl radical (NH 2 Y•), supporting the inclusion of these Ys in the RT pathway. 12, 13 In addition, NH 2 Y• formation has allowed the characterization of a tight α2β2 complex 24 and provided insight into three distinct PCET steps within this pathway using multi-frequency and high-field electron paramagnetic resonance (HF EPR), 25 electron-nuclear double resonance (ENDOR), 26–28 and pulsed electron-electron double resonance (PELDOR) spectroscopies. 29 Despite the incorporation of the NH 2 Y thermodynamic sink into the >200 mV uphill RT pathway (Figure 2), the NH 2 Y-RNRs retained steady state activities of 3 – 5% of the wild type (wt) reaction rate.…”

A Hot Oxidant, 3-NO2Y122 Radical, Unmasks Conformational Gating in Ribonucleotide Reductase

“…[β- 2 H 2 ]NO 2 Y was prepared by nitration of [β- 2 H 2 ]Y (98 atom% 2 H)33. The deuterium incorporation was >96% based on 1 H NMR analysis.…”

Exploring Autoionization and Photoinduced Proton-Coupled Electron Transfer Pathways of Phenol in Aqueous Solution

“…A PCET pathway is understood to involve the concerted transfer of a proton and electron, 12,13 rather than a stepwise transfer, and is recognized as an important mechanism in the functioning of many biological systems. 14,15 To date, electronic structure calculations have only examined small phenol-water clusters, revealing that direct photoexcitation to the 1 πσ* state leads to a net transfer of an H atom to the water cluster. 16,17 Here we report the use of ultrafast transient absorption (TA) spectroscopy methods to investigate the excited-state dynamics of phenol-h 6 (in H 2 O) and its phenol-d 1 isotopologue (in D 2 O) using a femtosecond UV pump excitation, and a broadband white light super-continuum probe that spans the electronic absorption of the phenoxyl radical and solvated electron species.…”