ENDOR Spectroscopy and DFT Calculations: Evidence for the Hydrogen-Bond Network Within α2 in the PCET of E. coli Ribonucleotide Reductase

Abstract

E.coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y122•) cofactor required to generate a thiyl radical (C439•) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y122 ⇆ W48[?] ⇆ Y356 in β2, across the subunit interface to Y731⇆ Y730 ⇆ C439 in α2. Within α2 a co-linear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH2Y) replaced Y730 in α2 and this mutant was incubated with β2, CDP and ATP to generate a (NH2Y730•) in D2O. [2H]-Electron-nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C439 and Y731. A third proton was assigned to a water molecule in close proximity (2.2 Å O-H---O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH2Y730• are consistent with the combined effect of the hydrogen bonds to Cys439 and Tyr731, both nearly perpendicular to the ring plane of NH2Y730. The results provide the first experimental evidence for the hydrogen bond network between the pathway residues in α2 of the active RNR complex, for which no structural data is available.

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

“…However, in our subsequent studies, DFT calculations on large cluster models that were generated from the crystal structure of NH 2 Y 730 –α2 (PDB 2XO4) suggested an almost planar NH 2 conformation in NH 2 Y 730 • and NH 2 Y 731 •. 26, 27 Thus, we decided to re-examine the NH 2 conformation in NH 2 Y 731 •-RNR.…”

“…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.…”

“…Our original multi-frequency EPR analysis of NH 2 Y 730 •-RNR 25 was interpreted to favor a pyramidal (RNH 2 Y•) structure, whereas later quantum chemical calculations on a geometry optimized structure of NH 2 Y 730 •-α2 and NH 2 Y 731 •-α2 predicted an almost planar conformation of the trapped NH 2 Y•s in the protein environment of α2. 26, 27 To obtain a direct measurement of the amino protons hyperfine coupling (hfc) and their orientations, we synthesized D 6 -NH 2 Y 731 -α2 and generated the D 6 -NH 2 Y 731 •-α2 in the presence of β2, substrate, and effector. This strategy permits a distinction between the resonances of the amino protons and all resonances of the internal 1 Hs in NH 2 Y•.…”

Cross-polarisation edited ENDOR

“…here [i-j] equal [1][2][3] and [2][3][4] for the {e-n} spin system and [1][2][3][4][5], [2][3][4][5][6], [3][4][5][6][7] and [4][5][6][7][8] for the {e-n 1 -n 2 } spin system. Angle ϕ [i−j ] corresponds to the projection of the effective field in the [i-j] electron manifold on the x-y plane.…”

“…Here, not only orientational selectivity but also increased resolution in the nuclear Larmor frequencies and suppression of second-order effects substantially simplify analysis and interpretation of hyperfine spectra. Representative examples for the application of ENDOR have been the elucidation of the ligand sphere of metal centres or the detection of hydrogen bond interactions in proteins [7][8][9][10]. Nevertheless, one substantial drawback of the currently used ENDOR techniques is the small ENDOR effect as compared to the available EPR signal.…”