¹⁹F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α₂β₂ enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β₂, nucleotide reduction in α₂. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y₃₅₆(β) and Y₇₃₁(α) across the α/β interface. Using 2,3,5-F₃Y₁₂₂-β₂ with 3,5-F₂Y₇₃₁-α₂, GDP (substrate) and TTP (allosteric effector), a Y₃₅₆^• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron–electron double resonance spectroscopies. 94 GHz ¹⁹F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y₃₅₆^• and the ¹⁹F nuclei of 3,5-F₂Y₇₃₁ in this RNR mutant. Similar experiments with the double mutant E₅₂Q/F₃Y₁₂₂-β₂ were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F₂Y₇₃₁. Remarkably, one conformation is consistent with 3,5-F₂Y₇₃₁ within the H-bond distance to Y₃₅₆^•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y₃₅₆ and Y₇₃₁. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

1. Introduction

Ribonucleotide reductases (RNRs) catalyze the conversion of four nucleoside di- or triphosphates (ND(T)Ps) to deoxyribonucleoside di- or triphosphates (dND(T)Ps) in all organisms (Figure 1).¹⁻³ RNRs are highly regulated enzymes playing an important role in controlling the ratio and relative amounts of dNTPs essential for the fidelity of DNA replication and repair. Imbalance in dNTP pools results in genomic instability and leads to disease states.⁴⁻⁶ RNRs’ essential role has made them targets for cancer and, more recently, antibiotic therapeutics.⁶⁻¹²

Figure 1.

Reduction of NDPs to dNDPs catalyzed by Escherichia coli class Ia RNR. The reduction is initiated by a thiyl radical (C₄₃₉^•), and the reducing equivalents are provided by the oxidation of C₂₂₅ and C₄₆₂ to a disulfide. Multiple turnovers require a redoxin reducing system such as thioredoxin (TR), thioredoxin reductase (TRR), and nicotinamide adenine dinucleotide phosphate (NADPH).

The E. coli class Ia RNR, a prototype model system for human RNR,⁶ is composed of two subunits, α¹³ and β,¹⁴ both required for activity. Based on their α₂ and β₂ structures, Uhlin and Eklund proposed a symmetrical α₂β₂ docking model (Figure 2A) for active RNR, which has played a central role in the experimental design.¹³ The model for substrate activation and chemistry requires that the diferric tyrosyl radical (Y₁₂₂^•) cofactor located in β₂ oxidizes C₄₃₉ to a thiyl radical in the active site of α₂, which, in turn, initiates NDP reduction (Figures 1 and 2C). Thiyl radical formation is proposed to occur by a radical transfer (RT) pathway, which involves five or six radical intermediates (Figure 2C),¹⁵ each generated by proton-coupled electron-transfer (PCET) steps.¹⁶⁻¹⁹

Figure 2.

Docking model¹³ (A) and cryo-EM structure³⁸ (B) of the α₂β₂ complex of E. coli class Ia RNR and the proposed RT pathway, (C) and (D), respectively. (A) The docking model based on the shape complementarity of subunits α₂¹³ and β₂.¹⁴ (B) Cryo-EM structure of an α₂β₂ complex of RNR generated when E₅₂Q/F₃Y₁₂₂-β₂, wt-α₂ GDP (substrate) and TTP (effector) were quenched at 50 s (pdb code: 6W4X).³⁸ Asymmetry of the complex is indicated by α′β′ (disordered pair) and αβ (ordered pair). (C) The proposed forward RT pathway based on many experiments.^{20−27,30−33} W₄₈ is shown in parentheses as there currently is no direct evidence for its involvement. The red and blue double arrows describe electron and proton transfers, respectively. Evidence for the bold water molecules has been reported recently.^27,28 (D) An intact RT pathway within αβ including Y₃₅₆ and its position relative to Y₇₃₁ is visible for the first time in the cryo-EM structure.³⁸ Distances between RT residues are indicated; the ¹⁹F atoms of 2,3,5-F₃Y₁₂₂ present in the cryo-EM structure have been omitted. Interfacial residue Q₅₂ (E₅₂ in wt-RNR) is included as it was important for stabilizing the α₂β₂ complex in the cryo-EM experiment.

Central for developing this model has been the ability to replace pathway Ys site-selectively with unnatural amino acids (UAAs) that have allowed the generation and thermodynamic trapping of pathway radical intermediates. The tyrosyl radicals (Y^•s) were studied by a suite of multifrequency electron paramagnetic resonance (EPR)²⁰⁻³⁰ methods as well as by transient absorption spectroscopic methods using photo-β₂ RNRs.³⁰⁻³⁴

Despite much insight into nature’s design for radical initiation in RNRs, elucidating the molecular basis for the RT across the α/β subunit interface has been hampered by the lack of structural information about the C-terminal tail of all βs (residues 341–375 in E. coli RNR), essential for α/β subunit interaction.³⁵⁻³⁷ The location of Y₃₅₆ in the RT pathway within this tail was thus unknown. Recently, a near-atomic resolution cryo-EM structure of a trapped α₂β₂E. coli complex was obtained (Figure 2B).³⁸ It was generated from the incubation of a double mutant of β₂, E₅₂Q/F₃Y₁₂₂-β₂, with wt-α₂, substrate (GDP), and allosteric effector (TTP) with freeze-quenching at 50 s. The 2,3,5-F₃Y₁₂₂ substitution allowed the generation of one dGDP product and accumulation of one pathway radical at Y₃₅₆^•. The E₅₂Q mutation was important for successfully trapping the α₂β₂ complex. The E₅₂ residue resides at the α/β-interface and is essential for activity, enabling proton release during Y₃₅₆ oxidation in the RT.^33,39

The cryo-EM structure (Figure 2B) revealed an asymmetric α₂β₂ complex, consistent with earlier results.^37,40 It also revealed the residues in the C-terminal tail of β (341–375) in an ordered αβ pair, the intact RT pathway including the location of Y₃₅₆ and its location relative to Y₇₃₁(α) (Figure 2D) for the first time. The entire C-terminal tail in α′/β′, where chemistry has occurred and Y₃₅₆^• is supposedly trapped, remains disordered.

The importance of Y₃₅₆ during RT has been established by many different methods that often led to the detection of the Y₃₅₆^• intermediate. Recent studies to identify the proton acceptor during its oxidation in forward RT revealed that the most reasonable candidates, E₅₂(β) and E₃₅₀(β), both conserved and essential,^36,39,41 are unlikely to be the ultimate acceptors.^33,34,42 These residues are located at ∼7 Å (E₅₂) and ∼14 Å (E₃₅₀) distances from the phenol-oxygen atom of Y₃₅₆ in the ordered αβ pair of the cryo-EM structure,³⁸ too far for direct proton or H atom transfer with Y₃₅₆.⁴³ A variety of ¹H and ¹⁷O high-frequency electron-nuclear double resonance (ENDOR) experiments on Y₃₅₆^•,^27,28 kinetic studies using RNRs with F_nY₃₅₆³³ and a photo-oxidant appended to the C₃₅₅ mutant of β, and pH studies of Y₃₅₆^• formation using F₂Y₃₅₆⁴² all support the interaction of Y₃₅₆^• with water (Figure 2C).

Efforts to understand the residues involved in managing the proton to support the PCET between Y₃₅₆ and Y₇₃₁ across the α/β interface have been less successful. The cryo-EM structure shows an O–O distance between Y₃₅₆ and Y₇₃₁ of ∼8 Å in ordered αβ, with Y₇₃₁ in its unusual stacked conformation with Y₇₃₀ as in previous X-ray structures of α₂ alone.¹³ While a number of pulsed electron double resonance (PELDOR) experiments⁶ revealed sharp distance distributions consistent with little Y₃₅₆^• flexibility, several different experiments reported the mobility of Y₇₃₁. In a crystal structure of NH₂Y₇₃₀-α₂ alone, Y₇₃₁ was found in a conformation where it is flipped away from the stacked conformation with NH₂Y₇₃₀.⁴⁴ PELDOR studies on a double mutant R₄₁₁A-NH₂Y₇₃₁-α₂ under turnover conditions revealed a conformational change of 3 Å in trapped NH₂Y₇₃₁^•, consistent with a flipping toward the α/β interface.²⁶ Subsequent studies using photo-β₂ with the same α₂ mutations revealed dynamic/rapid conformational changes of Y₇₃₁.³⁰ Another EPR study by Yokoyama et al. suggested the flipping of F₂Y₇₃₁^•,²³ which was trapped as a minority radical species in NO₂Y₁₂₂-β₂/F₂Y₇₃₁-α₂. Molecular dynamics (MD) simulations using the cryo-EM structure and the α/β interface in water also support the flexibility of Y₇₃₁,⁴⁵ with movement away from the stacked conformation with Y₇₃₀. The studies together support a model for PCET between Y₃₅₆^• and Y₇₃₁ across the α/β interface that could involve a movement of Y₇₃₁ toward the interface (Figure 2C), with consequences for their PCET chemistry. However, structural or spectroscopic evidence for interaction between Y₃₅₆^• and Y₇₃₁ has never been observed.

In this article, we use ¹⁹F–Y analogues introduced site-specifically into E. coli RNR, F₃Y₁₂₂-β₂ (or the double mutant E₅₂Q/F₃Y₁₂₂-β₂), incubated with 3,5-F₂Y₇₃₁-α₂, GDP, and TTP to generate and trap Y₃₅₆^•. F₂Y₇₃₁ was chosen for its symmetric ¹⁹F substitution pattern and minimally perturbed reduction potential relative to Y.^46,47 The Y₃₅₆^• location and identity are established using 34 GHz PELDOR and 263 GHz EPR spectroscopies, respectively. ¹⁹F ENDOR spectroscopy^48,49 at 94 GHz is used in an effort to determine the distances across the subunit interface between the trapped Y₃₅₆^•(β) and the ¹⁹F nuclei of F₂Y₇₃₁(α). The ENDOR spectra give unambiguous evidence for two conformations of F₂Y₇₃₁. One conformation is consistent with the structure observed by cryo-EM (ordered αβ pair). The second conformation indicates a flipping of F₂Y₇₃₁ toward Y₃₅₆^•. The results have important implications for the PCET mechanism across the α/β interface.

2. Materials and Methods

2.1. Preparation of RNR Mutants and Activity Assays

The RNR mutants F₃Y₁₂₂-β₂, E₅₂Q/F₃Y₁₂₂-β₂, F₂Y₇₃₁-α₂, and ¹⁷O–Y-wt-α₂ were expressed and purified, as previously described.^39,44,50 Activities of (E₅₂Q)F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ and wt-β₂/¹⁷O–Y-α₂ were determined using the spectrophotometric assay (Supporting Information (SI) 1, Table S1).⁵¹

2.2. EPR Sample Preparation

The Y₃₅₆^• intermediate was trapped by incubating a solution of F₂Y₇₃₁-α₂, GDP, and TTP in assay buffer (50 mM HEPES, 15 mM MgSO₄, 1 mM EDTA, pH 7.6) with F₃Y₁₂₂-β₂ or E₅₂Q/F₃Y₁₂₂-β₂ in assay buffer. Glycerol concentrations were optimized (Figure S1) and typically added to ∼20% of the final volume to prolong phase memory times T_M for PELDOR and ENDOR measurements. The final concentrations were ∼80 μM α₂β₂, ∼1 mM GDP, and ∼200 μM TTP. The reaction mixture was transferred to either 34 GHz EPR tubes (Q-band) (12 μL, 1.5 mm inner diameter (ID) Suprasil tube, Wilmad) or 94 GHz (W-band) tubes (4.4 μL, 0.7 mm ID clear fused quartz tubes) and quenched by freezing in liquid nitrogen at reaction times (T_Q) of 40–80 s (Q-band) or 35–55 s (W-band). A second set of samples were prepared with T_Q > 100 s. Two hundred and sixty-three GHz EPR samples were prepared in Suprasil capillaries (ID 0.2 mm, Vitrocom) without glycerol and quenched at T_Q = 15–20 s. All samples are summarized in SI 2, Table S2.

2.3. 263 GHz EPR Spectroscopy

High-frequency (HF) 263 GHz echo-detected EPR spectra were recorded with a commercial spectrometer, as previously reported.⁵² Details on the spectral acquisition are given in SI 3.

2.4. 34 GHz PELDOR Spectroscopy

Four-pulse PELDOR experiments^53,54 were performed at 34 GHz (Q-band) on a commercial Bruker ELEXSYS E580 EPR spectrometer, as previously reported.²⁷ An optimized temperature of 50 K was selected, where high sensitivity is achieved and unreacted F₃Y₁₂₂^• does not contribute to the spin echo under conditions used for data collection (SI 4.1–4.3). MW pulses were amplified by a pulsed 170 W TWT amplifier (Model 187Ka, Applied Systems Engineering) with typical pulse lengths of 14–16 ns for the pump π-pulse at the center of the overcoupled resonator. The observer frequency was set to −105 MHz from the dip center, leading to observer π-pulse lengths of 24–28 ns. The τ₁ value was 250 ns, and τ₂ values were optimized based on T_M measurements (SI 4.2). Shot repetition times were 4–6 ms. Time traces were recorded at three different observer positions (Figure S5) and their intensities were summed, reflecting their respective EPR signal strengths at that excitation position. Traces were analyzed with DeerAnalysis 2019,⁵⁵ using Tikhonov regularization (L-curve criterion for α parameter) and checked for consistency using neural network analysis.^56,57

2.5. 94 GHz ENDOR Spectroscopy

Pulsed EPR and ENDOR experiments at 94 GHz (W-band) were performed on a commercial Bruker ELEXSYS E680 EPR spectrometer, as previously described.²⁵ Using a 2 W MW amplifier, typical π/2 pulse lengths of 10–12 ns were achieved. EPR (echo-detected) spectra and signal contributions are illustrated in SI 5.1. Shot repetition times were optimized to 2–4 ms based on T₁ measurements (SI 5.2).

¹⁹F Mims ENDOR spectra of the Y₃₅₆^• were recorded using radio frequency (RF) pulses amplified by a 250 W RF amplifier (250A250A Amplifier Research). RF pulse lengths of 22 μs were used for ¹⁹F nuclei with ∼1.6 MHz couplings or 44 μs for couplings ≤∼250 kHz. RF pulse lengths were optimized using Rabi nutation experiments. Stochastic RF acquisition⁵⁸⁻⁶⁰ with 20 shots per point was used. To observe ¹⁹F couplings of different sizes, the adjustment of the interpulse delay τ in the Mims sequence was crucial. For couplings on the order of 1.6 MHz, two measurements with τ values of 236 and 266 ns were performed and summed subsequently (normalized to the number of scans) to attenuate the proton background. For smaller couplings, ≤∼250 kHz, τ was optimized to 620–622 ns (SI 5.3). ENDOR spectra were recorded at three different observer positions (Figure S8) and summed up with intensities reflecting their respective EPR signal strengths at that excitation position.

Data were collected at two temperatures. At 50 K, ENDOR sensitivity was higher than that at 80 K, where usually the signal of unreacted F₃Y₁₂₂^• disappears due to faster relaxation.²⁷ As a downside, at 50 K, the unreacted F₃Y₁₂₂^• contributed to the echo intensity of the Mims sequence at short interpulse delays τ. The contribution of F₃Y₁₂₂^• led to ¹⁹F ENDOR background signals, which had to be removed during data processing (SI 5.4). As a control for the background correction procedure, we repeated representative ¹⁹F ENDOR measurements at 80 K (SI 5.5–5.6) where no background of F₃Y₁₂₂^• was present. The results obtained at 50 and 80 K are fully consistent. In addition to the ¹⁹F background, broad, overlapping ¹H resonances associated with the 3,5-H atoms of Y₃₅₆²⁷ were identified by their changes observed with τ value changes and they were subtracted from the ¹⁹F spectra, as illustrated in SI 5.4.

¹⁷O ENDOR control experiments were performed using similar parameters described in our recent ¹⁷O ENDOR study²⁸ and are reported in SI 6.

2.6. Simulations of ENDOR Data

Mims ENDOR simulations of the Y₃₅₆^• were performed using EasySpin’s saffron routine.⁶¹ The g tensor was g_x = 2.0062, g_y = 2.0044, and g_z = 2.0022.²⁷ In the molecular frame, g_x is aligned along the C–O^• bond of Y₃₅₆^•, while g_y is perpendicular to this direction and in the plane of the aromatic ring. The strongly coupled β-proton of Y₃₅₆^• was included using previously reported hyperfine coupling (HFC) parameters.²⁷ For simulating the ¹⁹F ENDOR spectra with τ = 620–622 ns, the C3 and C5 protons²⁷ of Y₃₅₆^• were included. The ¹⁹F ENDOR line width parameter was simulated as 25 kHz for couplings below 0.5 MHz.⁴⁹ For larger couplings, a line width of 250 kHz was used. Chemical shift anisotropies were not resolved in the 94 GHz ¹⁹F ENDOR spectra.⁶²

2.7. Structural Models for ENDOR Analysis

Due to the large parameter space associated with the two Fs of F₂Y₇₃₁ and, as will become clear, their multiple side-chain conformations, a fitting routine that generates the most likely set of HFC parameters by minimizing residuals (rmsd) is not possible. We therefore used an approach similar to that described previously to analyze the PCET steps within α₂ using NH₂Y₇₃₁ and the X-ray structure of α₂ to position Y₇₃₀ and C₄₃₉.²⁵ In the present case, the small models were constructed starting from pdb 6W4X, the recent cryo-EM structure (resolution 3.3–5.5 Å).³⁸ Y₃₅₆ from β and Y₇₃₁ and Y₇₃₀ from α were extracted from the ordered α/β pair (Figure 2B,D). ¹⁹F atoms at C3 and C5 of Y₇₃₁ were introduced using PyMOL.⁶³ The peptide bonds connecting each tyrosine to their protein backbone were replaced by NHR and −CRO (Figure 3) groups, and their xyz coordinates were not changed compared to the cryo-EM structure. Density functional theory (DFT)-based, constrained geometry optimization using ORCA⁶⁴ resulted in the model structure S1 of the triad Y₃₅₆–F₂Y₇₃₁–Y₇₃₀. Further representative conformations of the triad were obtained by rotating around Cα/Cβ and Cβ-phenol bonds displacing the phenol side chains of Y₃₅₆ and F₂Y₇₃₁, as illustrated in Figure 3. Resulting models to fit the spectroscopic data are designated SX (X =1, 2, 3,...5) and are summarized in Tables S6 and S7 in SI 8. A water molecule binding to Y₃₅₆^• was also introduced into each model, with a binding geometry based on our previous studies (H-bond length ca. 1.8 Å, angle C4–O^•···H ca. 120°, C3–C4–O^•···H dihedral ca. 20°).^27,28 The effect of H-bonds on the spin density distribution,^65,66 further technical details on the DFT calculations, and the adaptation of the DFT-predicted parameters to the ENDOR simulations are described in the results section and summarized in SI 7. Contributions of the different conformations were assessed by rmsd analysis. Orientation-selective ¹⁹F spectra were then simulated using one set of parameters for all spectra.

Figure 3.

Models for the Y-triad. The black conformation corresponds to S1 but without the water molecule. The pink orientation of F₂Y₇₃₁ illustrates a flipped conformation, and the green orientation of Y₃₅₆^• represents a repositioning of the radical toward F₂Y₇₃₁, used in models S2–S5. Atom positions of the backbone are from the cryo-EM structure within ≤∼0.5 Å. R₁–R₄ peptide chains have been replaced by H atoms in S1–S5. Red arrows indicate a rotation around a bond, and dashed arrows indicate small rotations (Table S6).

3. Results

3.1. Characterization of RNR Constructs Using Activity Measurements, High-Field EPR, and PELDOR

The first part of the investigation required examination of the new RNR constructs that contain the ¹⁹F labels in F₂Y₇₃₁. Steady-state activities are reported in Table S1. Spectrophotometric assays revealed a specific activity of 560 nmol/(mg·min) (ca. 7% of wt) for F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂, defined with respect to the mass of β₂ in the assay. In contrast, an activity of only 6 nmol/(mg·min), that is, the lower limit of detection, was measured for E₅₂Q/F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂. The latter finding was expected, as the E₅₂Q mutation disrupts steady-state activity.³⁹

Nevertheless, both constructs are capable of one turnover and allowed trapping of the intermediate Y₃₅₆^• for EPR samples during back-radical transfer.⁶⁷ Moreover, glycerol is required in the sample preparation to prolong spin relaxation in the EPR experiments. Thus, the glycerol content (v%) was also optimized based on its effect on RNR activity (SI 1) and a value of 20 v% was selected for almost all samples (SI 2, Table S2). We characterized the structure of the trapped radical in F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ and E₅₂Q/F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ by 263 GHz EPR (SI 3). In all quenched reaction mixtures, two radical species were observed (Figure S2). One contribution arose from the unreacted F₃Y₁₂₂^• and was readily identified by its large g_x value (2.0082) and its characteristic ¹⁹F HFC structure. After subtracting a reference spectrum of F₃Y₁₂₂^•, the spectrum of the intermediate became visible (Figure S3). This radical was identified as Y₃₅₆^• due to the characteristic low g_x value of 2.0062 (reference spectrum of Y₃₅₆^• is shown in Figures S2 and S3), as reported with F₃Y₁₂₂-β₂/wt-α₂.²⁷ The analysis of the HF-EPR spectra also revealed no other radical species.

PELDOR spectroscopy (34 GHz) was then used to measure the diagonal distance between Y₃₅₆^• in one αβ pair and F₃Y₁₂₂^• in the second one (Figure 4). The orientation-averaged time traces exhibit clear oscillations. Indistinguishable results were obtained for various sample preparation conditions (SI 4). For comparison, a time trace of F₃Y₁₂₂-β₂/wt-α₂ was also measured (Figure 4, green). Distance distributions with a single peak centered at 3.03 ± 0.02 nm (Figure 4) and a width (full width at half-maximum (FWHM); Table S4) of 0.09–0.14 nm were obtained for all samples. The observed distance is typical for F₃Y₁₂₂^•–Y₃₅₆^• pairs.^6,27 From PELDOR and HF-EPR, we conclude that Y₃₅₆^• is the observed radical, as previously characterized using wt-α₂ for incubation.²⁷

Figure 4.

Orientation-averaged 34 GHz PELDOR time traces of F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ (∼80 μM, T_Q = 77 s, blue line), E₅₂Q/F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ (∼80 μM, T_Q = 44 s, red), and F₃Y₁₂₂-β₂/wt-α₂ (green) along with fits (dotted lines). Distance distributions are shown as the inset. A cartoon illustrates the assignment of distance peaks to radical pairs. A symmetric representation was chosen as the experiments reported herein do not inform about the asymmetry in the protein complex.

It is interesting to consider the observed distance within the framework of the new cryo-EM structure.³⁸ The detected radical intermediate (Y₃₅₆^•) is thought to be produced during reverse RT in the first turnover.⁶⁷ If the first turnover was occurring for instance in the α′β′ pair, see the notation from the cryo-EM structure (Figure 2B), then the observed PELDOR distance should be between Y₃₅₆^•(β′) and F₃Y₁₂₂^•(β). However, in the cryo-EM structure, the C-terminal β′ tail is disordered at the interface, indicating that the trapped state might be different under the conditions of the EPR experiments. Because of the disorder, the distance between F₃Y₁₂₂^•(β) and Y₃₅₆^•(β′) cannot be measured in the cryo-EM structure. If we consider the opposite diagonal distance, i.e., between the centroids⁶⁸ of the Tyr-O, C1, C3, and C5 atoms of F₃Y₁₂₂^• in β′ and Y₃₅₆^• in β, then the PELDOR distance of 3.0 nm is in agreement with this structure. We note that many such distances have been measured with other constructs.⁶ All give a sharp 3 nm distance feature, suggesting that the Y₃₅₆^• conformation is constrained. Our model for half-site RNR reactivity¹⁵ requires that the complex interconverts to allow for alternating PCET in αβ and α′β′. When the Y₃₅₆^• is trapped, the interconversion is slow. The kinetics of this structural interconversion and the mechanism of switching remain to be established but are likely to be critical for comparing results from different experimental setups.

3.2. Distance Measurements across the RNR α/β Interface Using 94 GHz ¹⁹F ENDOR

3.2.1. ¹⁹F ENDOR Detects Y₃₅₆^•–¹⁹F₂Y₇₃₁ Distances

¹⁹F ENDOR spectra of Y₃₅₆^• in F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ (black) and E₅₂Q/F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ (red) were obtained after summing three background-corrected, orientation-selective spectra in the range of ±4 MHz around the ¹⁹F Larmor frequency ν₀(¹⁹F) (Figure 5A). When using short τ values (236 and 266 ns), prominent resonances are observed at ±∼0.8 MHz in both samples. These resonances are attributed to one ¹⁹F nucleus, F_a, with a peak separation of ∼1.6 ± 0.1 MHz (purple, dashed lines). Additionally, sharp features are observed in a ±250 kHz region around ν₀(¹⁹F). These resonances were investigated using a larger τ value of 620 ns, which enhances the sensitivity for smaller couplings (Figure 5B).⁴⁹ For both samples, the spectra in Figure 5B can be interpreted as a superposition of two Pake patterns contributed by two ¹⁹F nuclei, designated as F_b and F_c. Pake patterns result from purely dipolar coupling and allow assignment of the corresponding dipolar HFC T by reading off the splitting between the sharp, central peaks: T_b = 250 ± 15 kHz (cyan, dashed lines) and T_c = 150 ± 15 kHz (green, dashed lines). These peaks are contributed by molecules in which the ¹⁹F-radical interspin vector is perpendicular to the external magnetic field B₀. Using the point-dipole approximation (eq 1)⁴⁹

1

we can estimate interspin distances of R_b = 6.7 ± 0.2 Å and R_c = 7.9 ± 0.3 Å, with the centroid of the O, C1, C3, and C5 atoms of Y₃₅₆^• as a point of reference.⁶⁸ Aside from the central peaks, Pake patterns are also characterized by shoulders appearing at twice the coupling strength (2·T = T_∥). These features are contributed by molecules with interspin vectors parallel to B₀. The dipolar approximation does not apply for the stronger coupling T_a due to the shorter distance, <5 Å.⁴⁹

Figure 5.

94 GHz ¹⁹F Mims ENDOR spectra of F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ (80 μM, T_Q = 50 s, black lines) and E₅₂Q/F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ (80 μM, T_Q = 35 s, red lines) at T = 50 K. Spectra in panels (A) and (B) were obtained by adding three orientation-selective spectra. (A) Measurement with short τ values (∼250 ns). (B) Measurement with larger τ values (∼620 ns). (C) Orientation-selective spectra with B₀ a∥ g_x and τ = 620 ns after data point smoothing with the Savitzky–Golay filter (full lines). Original data are shown as dotted lines. Measurement time per spectrum is 30–40 h (A) and 50–60 h (B). Analysis of the spectra in panels (A)–(C) requires consideration of four nuclei ¹⁹F_a–¹⁹F_d, as marked by arrows and colored dashed lines.

The observation of three distinct ¹⁹F resonances in Figure 5A,B requires at least two conformations of F₂Y₇₃₁. Since each conformation contributes two ¹⁹F–Y₃₅₆^• spin pairs, a fourth set of resonances (F_d) is expected but not clearly resolved in the spectra obtained by summing up three orientation-selective measurements. An indication for coupling to a fourth nucleus F_d was provided by the orientation-selective measurements with B₀ aligned along g_x (Figure 5C). Here, strong selectivity for the parallel components of F_b and F_c was observed. In addition, shoulders on the inside of the two most prominent features are observed, which suggest the parallel coupling of the fourth atom F_d. Further analysis of the orientation-selective spectra is discussed below and will confirm this assignment.

Interestingly, the size of the observed HFCs (peak positions) is conserved in both F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ and E₅₂Q/F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ mutants, but the spectrum of E₅₂Q/F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ in Figure 5A appears broader, suggesting more heterogeneity in this mutant.

3.2.2. Examination of Structural Models of the Triad Y₇₃₀–F₂Y₇₃₁–Y₃₅₆^•

To rationalize the ¹⁹F ENDOR spectra, structural models of the tyrosine triad were built (Section 2.7 and Figure 3) and the DFT-predicted ¹⁹F HFCs were compared with the experimental values in Figure 5. The starting point for modeling is the cryo-EM structure.³⁸ Model S1 (Figure 3, black) is identical to this structure, with two ¹⁹F nuclei replacing the 3,5-H atoms in Y₇₃₁. This structure results in HFCs of 65 kHz and 114 kHz (see also SI 8, Table S8), the latter approaching but not quite matching the 150 kHz indicated for F_c in Figure 5B given DFT uncertainties up to 20%. The 65 kHz coupling could potentially be attributed to the fourth ¹⁹F nucleus, F_d.

To increase the coupling strength in S1, either the position of F₂Y₇₃₁ or of Y₃₅₆^• had to be readjusted for the spin centers to come closer. An increase of T_c from 114 to ∼150 kHz for F_c would require reducing the interspin distance by roughly 1 Å based on eq 1. To maintain the stacked arrangement of F₂Y₇₃₁ and Y₇₃₀, observed in almost all available structures, we adjusted the position of O–Y₃₅₆^• by ca. 1 Å, which is still well within the resolution of the cryo-EM structure, as indicated in green color in Figure 3 (Table S6). This resulted in model S2, illustrated in Figure 6. We note that in model S2, as well as in all other models, a water molecule was introduced in the vicinity of Y₃₅₆^• (Section 2.7), the presence of which was reported earlier.^27,28 The H-bonding water molecule affects Y₃₅₆^•’s spin density distribution and, consequently, also the effective ¹⁹F-radical HFCs. As detailed in SI 7, the resulting geometrical changes are minor and amount to ca. 0.1–0.2 Å.

Figure 6.

Model S2, ¹⁹F–Y₃₅₆^• distances are indicated by dashed lines (centroid of Y₃₅₆^• as a point of reference). Fluorine, oxygen, and nitrogen atoms are in green, red, and blue, respectively. H₂O was included based on our previous results.^27,28

In S2, the ¹⁹F–Y₃₅₆^• distances are 9.8 and 8.4 Å, the latter consistent with the estimate for R_c based on the dipolar approximation (eq 1). DFT analysis of S2 predicts coupling constants of 85 and 153 kHz, reproducing the coupling of F_c in Figure 5B within the estimated uncertainty. The 85 kHz coupling could be attributed to F_d. When the triad shown in S2 is incorporated back into the cryo-EM structure, the position of Y₃₅₆^• was found to fulfill the PELDOR diagonal distance of 3.0 nm (Figure 4 and Table S7).

Nevertheless, it is clear that neither model S2 nor reorienting the ring plane of F₂Y₇₃₁ (model S3, Figure S16) is able to reproduce the observed strong HFCs of F_a.

We therefore examined the possibility that a second conformation between the interfacial Ys might result in a second pair of stronger ¹⁹F HFCs. This proposal is reasonable based on previous evidence from different types of experiments that Y₇₃₁ can flip.^{23,26,30,44,45} A small model based on the flipped Y-dyad taken from the X-ray structure of NH₂Y₇₃₀-α₂⁴⁴ (without β₂) could not be placed into the cryo-EM structure using pair fitting (in PyMOL) of the ring atoms to superimpose the Y₇₃₀ side chains since clashes resulted (SI 8, Figure S17). This is in principle expected because this structure is missing the β subunit, which provides structural constraints. We thus focused on αβ and returned to model S2, adjusted the dihedral angles around Cα–Cβ and N–Cα of Y₇₃₁ (Table S6), until the DFT-predicted HFC couplings reached the range of the experimental values for F_a and F_b. Representative structures that fulfilled the ¹⁹F HFCs are shown as models S4 and S5 (Figure 7), in which the fluorophenol groups are flipped by about 50–70° toward the subunit interface.

Figure 7.

Models S4 (A) and S5 (B). (A) Model S4 (fluorine, oxygen, and nitrogen atoms in green, red, and blue, respectively). H-bond lengths and the ¹⁹F-centroid (Y₃₅₆^•, yellow sphere) distances are indicated. (B) Model S5 (cyan sticks, colors as in panel A). (C and D) Top view of the models shown in panels (A) and (B). In panels (C) and (D), the g tensor of Y₃₅₆^• is indicated along with the parallel component of the dipolar HFC tensor of the distal ¹⁹F nucleus F_b.

In S4 (Figure 7A,C), the ¹⁹F nuclei reside at distances of 4.1 and 6.8 Å from the centroid of Y₃₅₆^•. For the proximal ¹⁹F atom (F_a), DFT predicts a dipolar coupling constant T_a of ∼1.0 MHz and a negative, isotropic coupling constant a_iso,a of −0.8 MHz. This combination leads to a splitting of ∼1.8 MHz for S4, similar to the ∼1.6 MHz observed experimentally for F_a (Figure 5A). The larger of the two ¹⁹F-radical distances in S4 agrees well with the estimate for R_b, yielding a coupling constant T_b of 254 kHz, in agreement with the resonances of F_b (Figure 5B).

In a second model with a flipped Y₇₃₁ (S5, Figure 7B,D), a distinct orientation of Y₇₃₁ and Y₃₅₆ was considered to account for orientation selection (see also next section). In S5, the ¹⁹F–Y₃₅₆^• distances are 4.6 and 7.3 Å. The interspin vector from the distal F_b to the centroid of Y₃₅₆^• is nearly parallel to the direction of g_x (Figure 7D) and distinct from S4 (Figure 7C). It has a DFT-derived HFC of T_b = 246 kHz. For the proximal ¹⁹F nucleus F_a, a dipolar coupling constant of T_a ≈ 0.8 MHz with a negative isotropic coupling constants a_iso,a of ca −1.0 MHz is predicted and leads to an expected peak separation of ∼1.8 MHz as in S4.

A comparison of DFT-predicted HFCs from all models, S1–S5, and the experimental values is shown in Figure 8. More details on geometrical parameters of the five models are summarized in Table S7. We note that the combination of S2 with either S4 or S5 could satisfy the experimentally observed peak separations in Figure 5.

Figure 8.

Comparison of experimentally observed peak separation from Figures 5 and 9 (purple (F_a), cyan (F_b), green (F_c), and brown (F_d) shadings indicate the range of uncertainty) with DFT-predicted peak positions (black squares) for models S1–S5. For the DFT values, an error of ±20% (F_a, this nucleus exhibits isotropic and anisotropic coupling) or ±10% (F_b–F_d, these nuclei show purely dipolar coupling) is estimated.

Finally, both S4 and S5, when integrated back into the framework of the cryo-EM structure,³⁸ give centroid–centroid distances between F₂Y₇₃₁ in αβ and F₃Y₁₂₂^• in α′β′ of 35.0 and 35.5 Å, respectively, both very similar to the constraints measured in our previous PELDOR experiments.²⁶

3.2.3. Spectral Simulations Including a Superposition of Stacked and Flipped Y₇₃₁ Conformations

The DFT analysis indicated that it is possible to find mutual conformations of F₂Y₇₃₁ and Y₃₅₆, which individually satisfy some observed ¹⁹F–Y₃₅₆^• distances. To examine whether a superposition of these conformations can reproduce the ENDOR spectra, we also considered the orientation-selected ENDOR spectra, which pose additional constraints with respect to the sum spectra of Figure 5.

Representative orientation-selected spectra, corresponding to the black sum spectra of Figure 5, are displayed in Figure 9. In the small coupling region (Figure 9B), we observe that T_∥(F_b) appears enhanced at g_x, suggesting an orientation of the F_b dipolar tensor parallel to g_x. Therefore, a structure similar to S5 likely describes the data better than S4, as illustrated in Figure 7C,D, where the orientation of the dipolar vector with respect to g_x is displayed.

Figure 9.

94 GHz ¹⁹F Mims ENDOR spectra on F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ (80 μM, T_Q = 50 s, black lines) at T = 50 K. (A) Measurement with short τ values (∼250 ns). (B) Measurement with larger τ values (∼620 ns). Simulations including four different ¹⁹F atoms (F_a–F_d) are shown as blue lines and are based on S2 and S5 (Tables 1 and 2). Contributions of individual ¹⁹F atoms are shown as shaded areas: purple (F_a), cyan, (F_b), green (F_c), and brown (F_d).

Using these orientational constraints, global simulations of the orientation-selective ENDOR spectra based on models S2 and S5 were carried out with the DFT-predicted parameters listed in Table 1 and the ratio (i.e., the relative contribution of S2 and S5) varied until a minimum of residual could be found (SI 9). rmsd from these simulations for all samples amount to ca. 0.1 or 10% at the optimized ratios (Figure S18). We observed that the simulation of the large coupling F_a (Figure 9A) is not very sensitive to the weighting of S2 and S5. This is expected as, under those experimental conditions, the resonances of F_b–F_d are suppressed by the Mims blind spot in the center of the spectrum. Instead, the ratio F_b/F_c affects the simulations of the small coupling region, as can be seen in Figure 9B by the decomposition of the simulation into the individual contributions. We note that the obtained weighting of the flipped conformation slightly varies between samples from 18 to 33% within an error of 5% for each sample (Table 2). Therefore, we estimate that the flipped conformation represents on average 25 ± 10% of the molecular ensemble.

Table 1. Parameters Used for the ENDOR Simulations.

atom (model)	F–Y356•a [Å]	Ax, Ay, Azb [kHz]	aiso [kHz]
Fa (S5)	4.6	580, −1668, −1952	–1013
Fb (S5)	7.3	–246, −246, 492	0
Fc (S2)	8.4	–159, −159, 318	0
Fd (S2)	10.0	–83, −83, 166	0

^aDistances defined with respect to the centroid of Y₃₅₆^•, as shown in Figures 6 and 7B.

^bCoupling constants A_i consider the anisotropic and the isotropic coupling constants (T_i and a_iso, respectively): A_i = T_i + a_iso. Euler angles for relating the A to g tensors are reported in Table S8. An error of ±15 kHz was estimated for couplings <500 kHz, while an error of ±125 kHz is estimated for the 1.6 MHz coupling (ca. 50% of the ENDOR line width parameter in both cases).

Table 2. Ratios of the Stacked Model S2 and the Flipped Model S5 from ENDOR Simulations.

RNR mutant	TQ [s]	contribution of flipped (S5)a
F3Y122-β2/F2Y731-α2	50	33%
F3Y122-β2/F2Y731-α2	143	22%
E52Q/F3Y122-β2/F2Y731-α2	35	18%
E52Q/F3Y122-β2/F2Y731-α2	153	25%

^aEstimated error: ±5%; see Figure S18.

The representative best simulation for one sample F₃Y₁₂₂-β₂/F₂Y₇₃₁-α₂ is superimposed on the experimental data in Figure 9. Remarkably, the simulation of the orientation-selective spectrum at B₀ ∥ g_x captures the selectivity for T_∥(F_b) and T_∥(F_c) and also reproduces the shoulders on the inner side, which were tentatively assigned to F_d in the discussion of Figure 5C. Given the challenges of the simulation procedure, we find that the obtained simulation reproduces the experimental data very satisfactorily.

3.3. ¹⁷O ENDOR with (E₅₂Q)-F₃Y₁₂₂-β₂/¹⁷O–Y-wt-α₂

An independent effort was made to obtain experimental evidence for a flipped Y₇₃₁ conformation in the trapped complex. We investigated whether a ¹⁷O ENDOR signal might be observable with a sample prepared using uniformly labeled ¹⁷O–Y-wt-α₂ (¹⁷O in the phenol groups). This experiment was motivated by our recent successful observation of a ¹⁷O ENDOR signal from water H-bonded to Y₃₅₆^•.²⁸ DFT calculations predicted a ¹⁷O–Y₇₃₁–Y₃₅₆^• coupling of ∼0.5 MHz for the flipped structure S5, slightly smaller than observed for H-bonded ¹⁷OH₂ (0.7 MHz) (Table S9). We further considered issues that might make detection of this interaction more challenging. ¹⁷O has a lower gyromagnetic ratio than ¹⁹F (γ(¹⁹F)/γ(¹⁷O) ≈ 6.95) and its quadrupolar coupling may lead to signal broadening. In addition, the ¹⁷O–Y₇₃₁-α₂ is only 35–40%-labeled based on the available ¹⁷O–Y used during expression (SI 6). A reference ENDOR signal, with a comparable concentration of predicted ¹⁷O spins in close proximity to Y₃₅₆^• (i.e., ca 10–20 μM), is shown in Figure S15. Despite potential unexpected issues, we proceeded with the experiment as ¹⁷O should be a sensitive nucleus at short distances (≲3 Å) and the ¹⁷O–Y₇₃₁ coupling for the stacked conformation should not be detectable, allowing us to test the flipped Y₇₃₁ model. As shown in SI 6.2, we were not able to observe any ¹⁷O couplings in three independently prepared samples. We have considered several possible explanations for these observations that may be related either to the experiment or to the use of F_nY probes: (1) the ¹⁷O coupling might be smaller than the DFT prediction and not detectable; (2) F₂Y₇₃₁ could experience a different flipping ratio or rate of flipping relative to Y₇₃₁; (3) the F₃Y₁₂₂^• used to initiate radical transfer in the experiment is likely reduced to its phenolate, not phenol as with Y₁₂₂^•, and could play a role for the subunit interaction. These scenarios will be further discussed in the next section.

4. Discussion

In this paper, we report the use of 94 GHz ¹⁹F ENDOR spectroscopy, which has provided new insight into the chemistry of RT between Y₃₅₆(β) and Y₇₃₁(α) of E. coli RNR located at the subunit interface (Figure 2C,D). Success was possible using enzymes with site-specifically incorporated F_nYs: F₃Y₁₂₂-β₂ (or E₅₂Q/F₃Y₁₂₂-β₂) and F₂Y₇₃₁-α₂, which, when incubated with substrate (GDP) and effector (TTP), allowed trapping of the Y₃₅₆^• pathway radical in an “active” α₂β₂ complex during the reverse RT pathway process. PELDOR and HF-EPR analysis established the location of the trapped radical, and the double mutant provided a direct link to the recent cryo-EM structure.³⁸ The studies allowed measurement of the ¹⁹F–Y₇₃₁ hyperfine couplings to Y₃₅₆^•, which report on their interspin distances and provide interesting mechanistic implications.

Analysis of 94 GHz ¹⁹F ENDOR spectra of the Y₃₅₆^• required careful evaluation and subtraction of ¹⁹F signals associated with unreduced F₃Y₁₂₂^• and ¹H backgrounds. Nevertheless, comparison of the spectra acquired at 50 and 80 K allowed unambiguous assignment of three distinct couplings between F₂Y₇₃₁ and Y₃₅₆^•.

Construction of small models of the three Ys and their DFT-predicted ¹⁹F HFC couplings, ENDOR orientation selection, and spectral simulations indicated that the ¹⁹F spectra are consistent with a mixture of flipped and stacked conformations of F₂Y₇₃₁ with respect to Y₇₃₀, with flipped contributions of 25 ± 10% among the samples. While the flexibility of Y₇₃₁ has been reported previously, the present results provide the first evidence for a conformation, in which the two pathway residues are located at an O–O distance of ∼3 Å, with potentially important consequences for understanding the interfacial PCET step. The presence of both conformations simultaneously suggests that they are energetically similar and may exist in equilibrium.

A number of different types of experiments have previously reported multiple Y₇₃₁ conformations.^26,30 In one study, in which CDP/ATP was incubated with wt-β₂/R₄₁₁A-NH₂Y₇₃₁-α₂, an NH₂Y₇₃₁^• intermediate trapped in the forward RT was observed.²⁶ The flipping was detected by PELDOR spectroscopy by its unusual Y₃₅₆^•/NH₂Y₇₃₁^• distance. This distance, however, was only observed in conjunction with an additional mutation at α-R₄₁₁A. This residue sits in the α/β interface. In addition, transient absorption experiments in solution using the same α-R₄₁₁A mutation and a photo-oxidant indicated a k_PCET between Y₃₅₆F-photoβ₂ and Y₇₃₁ much faster than dNDP formation, ∼10⁴ s^–1 versus 1–10 s^–1.³⁰

On the other hand, neither in the cryo-EM structure with E₅₂Q/F₃Y₁₂₂-β₂ nor in the ¹⁷O ENDOR experiments, which both employed F₃Y₁₂₂^• and wt-α₂, was the flipped conformation of Y₇₃₁ observed. Thus, while the role of F₂Y₇₃₁ in potentiating flipping is still unclear, the F₃-phenolate generated at residue 122 during RT may not be the basis for a flipped Y₇₃₁ conformation. In addition, the conditions for freeze-quenching the cryo-EM and ENDOR samples are very distinct in terms of protein concentration and glycerol content. A protein concentration of ∼80 μM had to be used for EPR samples, exceeding physiological RNR concentrations (ca. 1 μM). At elevated protein concentrations, the formation of α₄β₄ complexes has been reported.^69,70 However, these complexes are incapable of producing Y₃₅₆^• and should not affect the analysis of EPR experiments, in which Y₃₅₆^• was observed selectively.

Overall, the complex interplay between Y₃₅₆(β), Y₇₃₁(α), R₄₁₁(α), and other residues at the subunit interface is likely to be crucial for regulating the communication between the two redox-active Ys across the α/β interface.

Inspecting the predicted HFC parameters of the phenolic proton of F₂Y₇₃₁ with respect to Y₃₅₆^• is another interesting source of information. The DFT calculations predicted HFCs of ∼6 MHz in models S4 and S5. It is important to rationalize this finding in the context of previous ¹H ENDOR studies on H-bond interactions to Y₃₅₆^•.²⁷ In those studies, a ¹H coupling in the range of 6 MHz was observed and assigned to one (or 2 equiv) H-bonded water molecule(s). The presence of the second water molecule was postulated to explain the unprecedented low g_x value of Y₃₅₆^•, i.e., 2.0062.²⁷ The sharp peaks observed in our recent 263 GHz ¹⁷O ENDOR experiments support the presence of only a single water molecule.²⁸ Given the similarity of coupling constants for the H-bonded protons for Y₇₃₁ from either model S4 or S5, the flipped conformation provides an explanation for the ¹H coupling consistent with these previous ¹H ENDOR data. To date, however, no ENDOR study has provided information on the interplay between stacked/flipped Y₇₃₁ and the water binding at Y₃₅₆^•, which may be a key feature to control PCET across the interface. Interestingly, no distribution of g_x values at Y₃₅₆^• is observed, indicating that the electrostatic environment is well defined and similar in both Y₇₃₁ conformations. A mechanism, by which Y₇₃₁ replaces a water molecule as a H-bond donor to Y₃₅₆^• upon flipping, could explain this finding.

4.1. Implication of Flipped Y₇₃₁ in PCET across α/β

Observation of flipped F₂Y₇₃₁ in close distance to Y₃₅₆^•, trapped in an active RNR complex, enables the examination of a mechanism for the PCET step between Y₃₅₆^• and Y₇₃₁ for the first time.

The current hypothesis for interfacial PCET involving water, as noted above, was based on the ENDOR studies and the H-bond to Y₃₅₆^• assigned to water.^27,28 Recent MD simulations⁴⁵ based on the cryo-EM structure supported the role of water first suggested by Nick et al.²⁷ The simulations additionally showed that water molecules can be present at the α/β interface including between Y₃₅₆ and Y₇₃₁, between Y₃₅₆ and β-E₅₂ (an interface residue), and support a pathway for water to escape to the bulk solvent.^38,45 Interestingly, MD also revealed an equilibrium between flipped and stacked conformation for Y₇₃₁, both populated at room temperature.⁴⁵ Nevertheless, the reported flipped Y₇₃₁ structure from the MD study still shows a long O–O distance to Y₃₅₆ (∼8 Å on average), precluding a direct interaction between the two Ys.⁴⁵

Thus, the mechanism of PCET between Y₃₅₆ and Y₇₃₁ (i.e., during reverse and forward RTs) remained to be resolved due to the long Y₃₅₆–Y₇₃₁ distance (∼8 Å) observed in the cryo-EM structure.³⁸ We note that the published cryo-EM structure and ENDOR data have distinct problems. The resolution of the cryo-EM structure was insufficient to resolve waters. The ENDOR studies only detected water in the first coordination sphere of Y₃₅₆^•, i.e., in a distance range of ∼3 Å.^27,28

The ¹⁹F ENDOR data presented here, despite the issues raised, provide evidence for close interaction between the two Ys across the subunit interface in an active RNR construct. In our ENDOR-derived model S5, the O–O distance between Y₃₅₆^•–Y₇₃₁ amounts to 3.0 ± 0.2 Å, with a similar value in the related model S4. This distance is within the range of the distances reported for the pathway pair C₄₃₉–Y₇₃₀ (O–S: 3.7 Å in the X-ray structure of α₂ versus 3.4 Å in α-NH₂Y₇₃₀)^13,44 as well as for the pair Y₇₃₀–Y₇₃₁ (O–O: 3.3 Å in α₂ versus 2.7 Å in α-NH₂Y₇₃₀).^13,44 For these pairs, independent quantum chemical calculations predicted a colinear PCET mechanism,^24,71,72 in which the electron and proton are transferred individually in one step from the same donor to the same acceptor, although a water-assisted PCET has been proposed and discussed for the C₄₃₉–Y₇₃₀ pair.⁷³ Recently, also an alternative, glutamate (E₆₂₃)-mediated proton transfer for the RT between Y₇₃₁ and Y₇₃₀, has been proposed based on MD simulations and QM/MM analysis.⁷⁴ A key conclusion from the latter study based on the analysis of E₆₂₃ was that forward and reverse RTs are different. Interestingly, our earlier large-scale DFT calculation on the pathway triad C₄₃₉–Y₇₃₀–Y₇₃₁ predicted that the coordination of a water molecule to Y₇₃₀^• can stabilize this radical intermediate and the transition states to the next pathway intermediates, Y₇₃₁^• and C₄₃₉^•.²⁴ Therefore, the calculation pointed to a functional role of water in PCET without its direct involvement as a proton donor or acceptor. Based on these considerations, we propose that our current results are consistent with a model of colinear PCET mechanism for the RT Y₃₅₆^•(β) – Y₇₃₁(α) ⇄ Y₃₅₆(β) – Y₇₃₁^•(α). This mechanism requires a conformational change of Y₇₃₁ during the long-range RT, as the next step (Y₇₃₁^•(α) – Y₇₃₀(α) ⇄ Y₇₃₁(α) – Y₇₃₀^•(α)) occurs in the stacked conformation of the Y₇₃₁/Y₇₃₀ pair.

5. Conclusions

Use of site-specifically incorporated unnatural amino acids and kinetic trapping in conjunction with high-field ENDOR, PELDOR, and EPR spectroscopies has given new insight into the PCET involving Y₃₅₆^•(β) and Y₇₃₁(α) across the RNR subunit interface. ¹⁹F ENDOR revealed two sets of hyperfine coupling constants for F₂Y₇₃₁ caused by the occurrence of two distinct conformations. One set of hyperfine couplings is consistent with a stacked Y₇₃₁ conformation at an ∼8 Å distance (O–O) to Y₃₅₆^•, as observed by cryo-EM. However, much larger ¹⁹F couplings revealed a second conformation, in which F₂Y₇₃₁ is flipped toward Y₃₅₆^• at a much shorter O–O distance of ∼3 Å. This distance is similar to distances between other Y pairs on the RT pathway in α, for which colinear PCET has been established.

These results reveal again the ability and importance of EPR spectroscopic methods and new experimental designs for the detection of multiple conformations in a biological machinery.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c02906.

• Measurement of RNR activity, EPR sample preparation, EPR experiments (263, 34, and 94 GHz spectroscopies including relaxation measurements, radical yield determination, and background corrections), ¹⁷O ENDOR data, modeling of the tyrosine triad, and ENDOR simulations (PDF)

Author Present Address

^¶ Frankfurt Cancer Institute, Goethe University Frankfurt, 60596 Frankfurt, Germany

Author Present Address

^◆ Department of Medicine II, Hematology/Oncology, University Hospital Frankfurt, 60590 Frankfurt, Germany

Author Present Address

^▼ Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

The authors acknowledge funding from the Max Planck Society, the German DFG priority program SPP 1601, the ERC-BIO-enMR grant (contract 101020262), a fellowship of the graduate school GGNB (A.K., program Physics of Biological and Complex Systems), and the NIH grants GM047274 (C.C.) and GM-29595 (J.S. and C.C.). Open access funded by Max Planck Society.

The authors declare no competing financial interest.

Notes

Original spectroscopic data associated with Figures 5 and 9 as well as all xyz coordinates of the model structures can be accessed via the open database Göttingen Research Online (https://doi.org/10.25625/YXHC63).