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 A from a tyrosyl radical (Y(122)*) within the beta2 subunit to a cysteine residue (C(439)) within the alpha2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y(122) --> W(48) --> Y(356) in beta2, across the subunit interface to Y(731) --> Y(730) --> C(439) in alpha2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in alpha2. Incubation of this mutant with beta2, substrate, and allosteric effector resulted in loss of the Y(122)* and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH(2)Y*). In the current study [(15)N]- and [(14)N]-NH(2)Y(730)* have been generated in H(2)O and D(2)O 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 NH(2)Y(730)*. The g tensor (g(x) = 2.0052, g(y) = 2.0042, g(z) = 2.0022), the orientation of the beta-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 NH(2) moiety are consistent with an intramolecular hydrogen bond within NH(2)Y(730)*. This analysis is an essential first step in using the detailed structure of NH(2)Y(730)* to formulate a model for a PCET mechanism within alpha2 and for use of NH(2)Y in other systems where transient Y*s participate in catalysis.

Figure 1

Proposed radical initiation pathway in E. coli class Ia RNR. Radical transfer occurs by orthogonal PCET within β2, where long-distance ET within the pathway is coupled to short-distance off-pathway PT, and by collinear PCET (or hydrogen atom transfer) within α2. Note that the position of Y₃₅₆ is structurally unknown.

Figure 2

Structure of NH₂Y₇₃₀•. (Top) Numbering scheme (inside numbers), axis system, and the intramolecular H-bond represented by dashed lines. (Bottom) NH₂Y₇₃₀• viewed along the C3-N and the phenol C2−C3 bond within the aromatic plane. Dihedral angles for the amino protons (left) and the Cβ-protons (middle) of NH₂Y₇₃₀• with respect to the phenol plane, which is indicated by dashed lines. (Right) Tetrahedral nature of the NH₂ moiety and the 5° tilt of the C3−N bond away from the aromatic plane, which is indicated by a bold line.

Figure 3

Normalized EPR spectra of isotopically substituted NH₂Y₇₃₀•. Spectra were acquired with Y₇₃₀NH₂Y-α2/β2 containing [¹⁴N]-NH₂Y or [¹⁵N]-NH₂Y in H₂O or D₂O assay buffer in the presence of CDP/ATP. The ticks on the deuterated traces indicate the positions of the quartet and triplet peaks with [¹⁴N]- and [¹⁵N]-NH₂Y₇₃₀•, respectively. See text for details.

Figure 4

Multifrequency EPR spectra of NH₂Y₇₃₀• in the presence of CDP/ATP in H₂O assay buffer. (A) 180 GHz spectrum at 6 K consisting of Y₁₂₂• and NH₂Y₇₃₀• signals. Inset: 180 GHz spectrum at 70 K containing only the NH₂Y₇₃₀• signal. (B) Spectrum of NH₂Y₇₃₀• at 9, 94, and 180 GHz, demonstrating the higher g resolution with increasing frequency. The spectrum at 180 GHz yields g values of g_x = 2.00520, g_y = 2.00420, and g_z = 2.00220. The error in these measurements was ±0.0001.

Figure 5

94 GHz pulsed derivative EPR spectra of NH₂Y₇₃₀• in H₂O (black trace) and D₂O (red trace). Experimental parameters: echo sequence: π/2 = 32 ns, τ = 248 ns (in H₂O), τ = 260 ns (in D₂O), π = 64 ns; t_rep = 5 ms; shots/point = 50; scans = 700; T = 70 K. The ticks on the deuterated trace indicate the positions of the quartet. See text for details.

Figure 6

Simulations (blue = SimFonia; green = EasySpin) of the first derivative EPR spectra (red) of [¹⁴N]-NH₂Y₇₃₀• and [¹⁵N]-NH₂Y₇₃₀• in D₂O assay buffer. (A) [¹⁴N]-NH₂Y₇₃₀• at 9 GHz. (B) [¹⁵N]-NH₂Y₇₃₀• at 9 GHz. (C) [¹⁴N]-NH₂Y₇₃₀• at 94 GHz. See Table 1 for parameters. The simulations obtained with EasySpin and the parameters of Table 1 are in agreement with those from SimFonia.

Figure 7

Simulations (blue = SimFonia; green = EasySpin) of the first derivative EPR spectra (red) of [¹⁴N]-NH₂Y₇₃₀• and [¹⁵N]-NH₂Y₇₃₀• in H₂O assay buffer. (A) [¹⁴N]-NH₂Y₇₃₀• at 94 GHz. (B) [¹⁴N]-NH₂Y₇₃₀• at 9 GHz. (C) [¹⁵N]-NH₂Y₇₃₀• at 9 GHz. See Table 1 for parameters. The simulations with EasySpin appear slightly broader due to the effect of forbidden transitions.

Figure 8

Model of the Y₇₃₁−NH₂Y₇₃₀−C₄₃₉ pathway in NH₂Y₇₃₀-α2. NH₂Y has been modeled into residue 730 of α2 using the coordinates from ref (14). The p_z orientation and C_β- and amino proton dihedral angles are based on results herein. The distance between the NH₁ and the phenol O is indicated. The distances between the essential residues around NH₂Y₇₃₀ are illustrated as discussed in the text.