Radical transfer in E. coli ribonucleotide reductase: a NH₂Y₇₃₁/R₄₁₁A-α mutant unmasks a new conformation of the pathway residue 731

Abstract

A new conformation of the E. coli RNR pathway residue 731 was trapped during long-range radical transfer across the αβ subunit interface.

Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase

“…ENDOR spectra for Y 731 NH 2 Y•, Y 730 NH 2 Y• and Y 731 NH 2 Y•′ have been previously recorded 31,36 and the corresponding HYSCORE spectra in Figures 2A, 2B and 2C, respectively, are consonant with these ENDOR results. The ENDOR data, in conjunction with DFT modelling, revealed the number of H-bonds at each respective NH 2 Y site and the associated hyperfine coupling (HFC) constants consistent with the collinear PCET pathway of α 2 .…”

“…The optical signature of NH 2 Y• ( λ max = 320 nm) is distinct from native (Y 122 •) or adventitiously generated Y radical ( λ max = 410 nm). 36,46 In the stopped flow experiments, mixing of Y 730 NH 2 Y-α 2 or Y 730 NH 2 Y′-α 2 resulted in biphasic transfer of the radical species from Y 122 (β) to Y 730 NH 2 Y(α) with rate constants analogous to previous measurements. 33,36 Fits of the data for Y 730 NH 2 Y• formation in Y 730 NH 2 Y-α 2 furnished apparent rate constants of 7.4(5) and 0.63(6) s −1 , representing 66% and 34% of the amplitude change respectively.…”

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

“…61 The g values of D 6 -NH 2 Y 731 • (2.0051, 2.0040, 2.0022) are best resolved at 263 GHz and are consistent with the values from our previous ND 2 Y studies. 25–28 Comparison of 94 GHz EPR spectra of H 6 -NH 2 Y• in D 2 O and D 6 -NH 2 Y• in H 2 O (Figure S11) clearly reveals the advantages of our deuteration approach. Use of D 6 -NH 2 Y• in H 2 O considerably simplifies the EPR spectra due to the absence of the two Cβ 1 H hf splitting and subtle hf structure from the amino protons becomes visible.…”

“…As described in our previous HF EPR studies, 25–28, 34 the spectrum of the trapped radical can be separated from that of stable Y 122 • by pulsed EPR at 80 K. The recorded 34, 94 and 263 GHz EPR spectra are shown in Figures S11 and S12, along with simulations for a planar NH 2 structure. The large hfcs of the two amino protons and the nitrogen nucleus dominate the D 6 -NH 2 Y 731 • EPR spectrum at 34 GHz (Figure S12).…”

“…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.…”