Pulsed Electron-electron Double Resonance Research Articles

Conformational Heterogeneity of β-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy.

Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of β-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd3+-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34GHz), the data analysis, and interpretation.

Nitrogen Substitutions Aggregation and Clustering in Diamonds as Revealed by High-Field Electron Paramagnetic Resonance.

Diamonds have been shown to be an excellent platform for quantum computing and quantum sensing applications. These applications are enabled by the presence of defects in the lattice, which are also known as color centers. The most common nitrogen-based defect in synthetic diamonds is the paramagnetic nitrogen substitution (P1) center. While the majority of quantum applications rely on nitrogen-vacancy (NV) centers, the properties of the latter are heavily influenced by the presence and the spatial distribution of the P1 centers. Hence, understanding the spatial distribution and mutual interactions of P1 centers is crucial for the successful development of diamond-based quantum devices. Unlike NV centers, P1 centers do not have a spin-dependent optical signature, and their spin-related properties, therefore, have to be detected and characterized using magnetic resonance methods. We show that using high-field (6.9 and 13.8 T) pulsed electron paramagnetic resonance (EPR) and dynamic nuclear polarization (DNP) experiments, we can distinguish and quantify three distinct populations of P1 centers: isolated P1 centers, weakly interacting ones, and exchange-coupled ones that are clustered together. While such clustering was suggested before, these clusters were never detected directly and unambiguously. Moreover, by using electron-electron double resonance (ELDOR) pump-probe experiments, we demonstrate that the latter clustered population does not exist in isolation but coexists with the more weakly interacting P1 centers throughout the diamond lattice. Its presence thus strongly affects the quantum properties of the diamond. We also show that the existence of this population can explain recent hyperpolarization results in type Ib high-pressure, high-temperature (HPHT) diamonds. We propose a combination of high-field pulsed EPR, ELDOR, and DNP as a tool for probing the aggregation state and interactions among different populations of nitrogen substitution centers.

P1 Center Electron Spin Clusters Are Prevalent in Type Ib Diamonds.

Understanding the spatial distribution of the P1 centers is crucial for diamond-based sensors and quantum devices. P1 centers serve as polarization sources for dynamic nuclear polarization (DNP) quantum sensing and play a significant role in the relaxation of nitrogen vacancy (NV) centers. Additionally, the distribution of NV centers correlates with the distribution of P1 centers, as NV centers are formed through the conversion of P1 centers. We utilized DNP and pulsed electron paramagnetic resonance (EPR) techniques that revealed strong clustering of a significant population of P1 centers that exhibit exchange coupling and produce asymmetric line shapes. The 13C DNP frequency profile at a high magnetic field revealed a pattern that requires an asymmetric EPR line shape of the P1 clusters with electron-electron (e-e) coupling strengths exceeding the 13C nuclear Larmor frequency. EPR and DNP characterization at high magnetic fields was necessary to resolve energy contributions from different e-e couplings. We employed a two-frequency pump-probe pulsed electron double resonance technique to show cross-talk between the isolated and clustered P1 centers. This finding implies that the clustered P1 centers affect all of the P1 populations. Direct observation of clustered P1 centers and their asymmetric line shape offers a novel and crucial insight into understanding magnetic noise sources for quantum information applications of diamonds and for designing diamond-based polarizing agents with optimized DNP efficiency for 13C and other nuclear spins of analytes. We propose that room temperature 13C DNP at a high field, achievable through straightforward modifications to existing solution-state NMR systems, is a potent tool for evaluating and controlling diamond defects.

PDSFit: PDS data analysis in the presence of orientation selectivity, g-anisotropy, and exchange coupling.

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS), encompassing techniques such as pulsed electron-electron double resonance (PELDOR or DEER) and relaxation-induced dipolar modulation enhancement (RIDME), is a valuable method in structural biology and materials science for obtaining nanometer-scale distance distributions between electron spin centers. An important aspect of PDS is the extraction of distance distributions from the measured time traces. Most software used for this PDS data analysis relies on simplifying assumptions, such as assuming isotropic g-factors of ~2 and neglecting orientation selectivity and exchange coupling. Here, the program PDSFit is introduced, which enables the analysis of PELDOR and RIDME time traces with or without orientation selectivity. It can be applied to spin systems consisting of up to two spin centers with anisotropic g-factors and to spin systems with exchange coupling. It employs a model-based fitting of the time traces using parametrized distance and angular distributions, and parametrized PDS background functions. The fitting procedure is followed by an error analysis for the optimized parameters of the distributions and backgrounds. Using five different experimental data sets published previously, the performance of PDSFit is tested and found to provide reliable solutions.

Simultaneous Localization of Two High Affinity Divalent Metal Ion Binding Sites in the Tetracycline RNA Aptamer with Mn2+-Based Pulsed Dipolar EPR Spectroscopy.

Mg2+ ions play an essential part in stabilizing the tertiary structure of nucleic acids. While the importance of these ions is well documented, their localization and elucidation of their role in the structure and dynamics of nucleic acids are often challenging. In this work, pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) was used to localize two high affinity divalent metal ion binding sites in the tetracycline RNA aptamer with high accuracy. For this purpose, the aptamer was labeled at different positions with a semirigid nitroxide spin label and diamagnetic Mg2+ was replaced with paramagnetic Mn2+, which did not alter the folding process or ligand binding. Out of the several divalent metal ion binding sites that are known from the crystal structure, two binding sites with high affinity were detected: one that is located at the ligand binding center and another at the J1/2 junction of the RNA.

Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis-Cu2+ (NTA) Motif.

Pulsed dipolar EPR spectroscopy (PDS) in combination with site-directed spin labeling is a powerful tool in structural biology. However, the commonly used spin labels are conjugated to biomolecules via rather long and flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label copper(II)-nitrilotriacetic acid [Cu2+ (NTA)] bound to two histidines (dHis) is rigid and yields narrow distance distributions, which can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDa Yersinia outer protein O (YopO) to decipher whether a previously experimentally observed bimodal distance distribution is due to two conformations of the biomolecule or of the flexible spin labels. Two different PDS experiments, that is, pulsed electron-electron double resonance (PELDOR aka DEER) and relaxation-induced dipolar modulation enhancement (RIDME), yield unimodal distance distribution with the dHis-Cu2+ (NTA) motif; this result suggests that the α-helical backbone of YopO adopts a single conformation in frozen solution. In addition, we show that the Cu2+ (NTA) label preferentially binds to the target double histidine (dHis) sites even in the presence of 22 competing native histidine residues. Our results therefore suggest that the generation of a His-null background is not required for this spin labeling methodology. Together these results highlight the value of the dHis-Cu2+ (NTA) motif in PDS experiments.

Insights into the protonation state and spin structure for the g = 2 multiline electron paramagnetic resonance signal of the oxygen-evolving complex.

In photosystem II (PSII), one-electron oxidation of the most stable oxidation state of the Mn4CaO5 cluster (S1) leads to formation of two distinct states, the open-cubane S2 conformation [Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV)] with low spin and the closed-cubane S2 conformation [Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III)] with high spin. In electron paramagnetic resonance (EPR) spectroscopy, the open-cubane S2 conformation exhibits a g = 2 multiline signal. However, its protonation state remains unclear. Here, we investigated the protonation state of the open-cubane S2 conformation by calculating exchange couplings in the presence of the PSII protein environment and simulating the pulsed electron-electron double resonance (PELDOR). When a ligand water molecule, which forms an H-bond with D1-Asp61 (W1), is deprotonated at dangling Mn4(IV), the first-exited energy (34 cm-1) in manifold spin excited states aligns with the observed value in temperature-dependent pulsed EPR analyses, and the PELDOR signal is best reproduced. Consequently, the g = 2 multiline signal observed in EPR corresponds to the open-cubane S2 conformation with the deprotonated W1 (OH-).

Inner Amino Acid Contacts Are Key Factors of Multistage Structural Rearrangements of DNA and Affect Substrate Specificity of Apurinic/Apyrimidinic Endonuclease APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most important enzymes in base excision repair. Studies on this enzyme have been conducted for a long time, but some aspects of its activity remain poorly understood. One such question concerns the mechanism of damaged-nucleotide recognition by the enzyme, and the answer could shed light on substrate specificity control in all enzymes of this class. In the present study, by pulsed electron-electron double resonance (DEER, also known as PELDOR) spectroscopy and pre-steady-state kinetic analysis along with wild-type (WT) APE1 from Danio rerio (zAPE1) or three mutants (carrying substitution N253G, A254G, or E260A), we aimed to elucidate the molecular events in the process of damage recognition. The data revealed that the zAPE1 mutant E260A has much higher activity toward DNA substrates containing 5,6-dihydro-2'-deoxyuridine (DHU), 2'-deoxyuridine (dU), alpha-2'-deoxyadenosine (αA), or 1,N6-ethenoadenosine (εA). Examination of conformational changes in DNA clearly revealed multistep DNA rearrangements during the formation of the catalytic complex. These structural rearrangements of DNA are directly associated with the capacity of damaged DNA for enzyme-induced bending and unwinding, which are required for eversion of the damaged nucleotide from the DNA duplex and for its placement into the active site of the enzyme. Taken together, the results experimentally prove the factors that control substrate specificity of the AP endonuclease zAPE1.

Darobactin B Stabilises a Lateral-Closed Conformation of the BAM Complex in E. coli Cells.

The β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.

Darobactin B Stabilises a Lateral-Closed Conformation of the BAM Complex in E. coli Cells.

The β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.

Quantitative analysis of sterol-modulated monomer–dimer equilibrium of the β1-adrenergic receptor by DEER spectroscopy

G protein-coupled receptors (GPCR) activate numerous intracellular signaling pathways. The oligomerization properties of GPCRs, and hence their cellular functions, may be modulated by various components within the cell membrane (such as the presence of cholesterol). Modulation may occur directly via specific interaction with the GPCR or indirectly by affecting the physical properties of the membrane. Here, we use pulsed Q-band double electron-electron resonance (DEER) spectroscopy to probe distances between R1 nitroxide spin labels attached to Cys163 and Cys344 of the β1-adrenergic receptor (β1AR) in n-dodecyl-β-D-maltoside micelles upon titration with two soluble cholesterol analogs, cholesteryl hemisuccinate (CHS) and sodium cholate. The former, like cholesterol, inserts itself into the lipid membrane, parallel to the phospholipid chains; the latter is aligned parallel to the surface of membranes. Global quantitative analysis of DEER echo curves upon titration of spin-labeled β1AR with CHS and sodium cholate reveal the following: CHS binds specifically to the β1AR monomer at a site close to the Cys163-R1 spin label with an equilibrium dissociation constant [Formula: see text]~1.4± 0.4mM. While no direct binding of sodium cholate to the β1AR receptor was observed by DEER, sodium cholate induces specific β1AR dimerization ([Formula: see text]~35± 6mM and a Hill coefficient n~ 2.5±0.4) with intersubunit contacts between transmembrane helices 1 and 2 and helix 8. Analysis of the DEER data obtained upon the addition of CHS to the β1AR dimer in the presence of excess cholate results in dimer dissociation with species occupancies as predicted from the individual KD values.

An improvement to capture and visualize the protein conformational ensembles in double electron-electron resonance measurements.

Over the past decades, pulsed ESR double electron-electron resonance (DEER) has emerged as an effective biophysical tool for revealing protein conformational ensembles. With a collection of numerous interspin distances by DEER, they can be utilized to reveal the dynamic structural ensembles as opposed to static structures from other techniques. In a continuing effort to improve the technique, we present a combined approach of experimental and simulation advances that allows one to capture and visualize protein conformational ensembles in the absence of cryoprotectant additives. First, we present a rapid freezing method using a flexible fused silica microcapillary, enabling DEER measurement without the use of cryoprotectants. Second, we present a Monte Carlo based algorithm that allows one to efficiently utilize the sparse DEER data for deriving ensemble structures of a target protein. The Bid protein, a highly glycerol-sensitive protein, is used as a model. We show that our rapid-freezing DEER method can capture the Bid ensembles in the absence of glycerol, which was not possible with the conventional DEER setup. With the newly developed algorithm, we can efficiently visualize the Bid conformational ensembles, saving a lot for performing advanced structural modeling. Our combined method advances DEER for capturing and visualizing protein conformational ensembles.

Gd3+-Trityl-Nitroxide Triple Labeling and Distance Measurements in the Heterooligomeric Cobalamin Transport Complex in the Native Lipid Bilayers.

Increased efforts are being made for observing proteins in their native environments. Pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) is a powerful tool for this purpose. Conventionally, PELDOR employs an identical spin pair, which limits the output to a single distance for monomeric samples. Here, we show that the Gd3+-trityl-nitroxide (NO) three-spin system is a versatile tool to study heterooligomeric membrane protein complexes, even within their native membrane. This allowed for an independent determination of four different distances (Gd3+-trityl, Gd3+-NO, trityl-NO, and Gd3+-Gd3+) within the same sample. We demonstrate the feasibility of this approach by observing sequential ligand binding and the dynamics of complex formation in the cobalamin transport system involving four components (cobalamin, BtuB, TonB, and BtuF). Our results reveal that TonB binding alone is sufficient to release cobalamin from BtuB in the native asymmetric bilayers. This approach provides a potential tool for the structural and quantitative analysis of dynamic protein-protein interactions in oligomeric complexes, even within their native surroundings.

Phosphorothioate-Based Site-Specific Labeling of Large RNAs for Structural and Dynamic Studies.

Pulsed electron-electron double resonance (PELDOR) spectroscopy, X-ray scattering interferometry (XSI), and single-molecule Förster resonance energy transfer (smFRET) are molecular rulers that provide inter- or intramolecular pair-wise distance distributions in the nanometer range, thus being ideally suitable for structural and dynamic studies of biomolecules including RNAs. The prerequisite for such applications requires site-specific labeling of biomolecules with spin labels, gold nanoparticles, and fluorescent tags, respectively. Recently, site-specific labeling of large RNAs has been achieved by a combination of transcription of an expanded genetic alphabet containing A-T/G-C base pairs and NaM-TPT3 unnatural base pair (UBP) with post-transcriptional modifications at UBP bases by click chemistry or amine-NHS ester reactions. However, due to the bulky sizes of functional groups or labeling probes used, such strategies might cause structural perturbation and decrease the accuracy of distance measurements. Here, we synthesize an α-thiophosphorylated variant of rTPT3TP (rTPT3αS), which allows for post-transcriptional site-specific labeling of large RNAs at the internal α-phosphate backbone via maleimide-modified probes. Subsequent PELDOR, XSI, and smFRET measurements result in narrower distance distributions than labeling at the TPT3 base. The presented strategy provides a new route to empower the molecular rulers for structural and dynamic studies of large RNA and its complex.

Combined Pulsed Electron Double Resonance EPR and Molecular Dynamics Investigations of Calmodulin Suggest Effects of Crowding Agents on Protein Structures.

Calmodulin (CaM) is a highly dynamic Ca2+-binding protein that exhibits large conformational changes upon binding Ca2+ and target proteins. Although it is accepted that CaM exists in an equilibrium of conformational states in the absence of target protein, the physiological relevance of an elongated helical linker region in the Ca2+-replete form has been highly debated. In this study, we use PELDOR (pulsed electron–electron double resonance) EPR measurements of a doubly spin-labeled CaM variant to assess the conformational states of CaM in the apo-, Ca2+-bound, and Ca2+ plus target peptide-bound states. Our findings are consistent with a three-state conformational model of CaM, showing a semi-open apo-state, a highly extended Ca2+-replete state, and a compact target protein-bound state. Molecular dynamics simulations suggest that the presence of glycerol, and potentially other molecular crowding agents, has a profound effect on the relative stability of the different conformational states. Differing experimental conditions may explain the discrepancies in the literature regarding the observed conformational state(s) of CaM, and our PELDOR measurements show good evidence for an extended conformation of Ca2+-replete CaM similar to the one observed in early X-ray crystal structures.

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Reveals Buffer-Modulated Cooperativity of Metal-Templated Protein Dimerization.

Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the noncovalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, pulse electron–electron double resonance and relaxation-induced dipolar modulation enhancement, were simultaneously employed to study the CuII-templated dimerization behavior of a model protein (Streptococcus sp. group G, protein G B1 domain) in both phosphate and Tris-HCl buffers. A cooperative binding model could simultaneously fit all data and demonstrate that the cooperativity of protein dimerization across α-helical double-histidine motifs in the presence of CuII is strongly modulated by the buffer, representing a platform for highly tunable buffer-switchable templated dimerization. Hence, PDS enriches the family of techniques for monitoring binding processes, supporting the development of novel strategies for bioengineering structures and stable architectures assembled by an initial metal-templated dimerization.

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modelling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology.

Solution-State Inter-Copper Distribution of Redox Partner-Linked Copper Nitrite Reductases: A Pulsed Electron-Electron Double Resonance Spectroscopy Study.

Copper nitrite reductases (CuNiRs) catalyze the reduction of nitrite to form nitric oxide. In recent years, new classes of redox partner linked CuNiRs have been isolated and characterized by crystallographic techniques. Solution-state biophysical studies have shed light on the complex catalytic mechanisms of these enzymes and implied that protein dynamics may play a role in CuNiR catalysis. To investigate the structural, dynamical, and functional relationship of these CuNiRs, we have used protein reverse engineering and pulsed electron–electron double resonance (PELDOR) spectroscopy to determine their solution-state inter-copper distributions. Data show the multidimensional conformational landscape of this family of enzymes and the role of tethering in catalysis. The importance of combining high-resolution crystallographic techniques and low-resolution solution-state approaches in determining the structures and mechanisms of metalloenzymes is emphasized by our approach.

Quantitative Agreement between Conformational Substates of Holo Calcium-Loaded Calmodulin Detected by Double Electron–Electron Resonance EPR and Predicted by Molecular Dynamics Simulations

Calcium-loaded calmodulin (CaM/4Ca2+) comprises two domains that undergo rigid body reorientation from a predominantly extended conformation to a compact one upon binding target peptides. A recent replica-exchange molecular dynamics (MD) simulation on holo CaM/4Ca2+ suggested the existence of distinct structural clusters (substates) along the path from extended to compact conformers in the absence of substrates. Here, we experimentally demonstrate the existence of CaM/4Ca2+ substates trapped in local minima by three freezing/annealing regimes (slow, 40 s; intermediate, 1.5 s; fast, 0.5 ms) using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure interdomain distances between nitroxide spin-labels positioned at A17C and A128C in the N- and C-terminal domains, respectively. The DEER echo curves were directly fit to population-optimized P(r) pairwise distance distributions calculated from the coordinates of the MD clusters and compact crystal structure. DEER data on fully deuterated CaM/4Ca2+ were acquired at multiple values of the second echo period (10-35 μs) and analyzed globally to eliminate instrumental and overfitting artifacts and ensure accurate populations, peak positions, and widths. The DEER data for all three freezing regimes are quantitatively accounted for within experimental error by 5-6 distinct conformers comprising a predominantly populated extended form (60-75%) and progressively more compact states whose populations decrease as the degree of compactness increases. The shortest interdomain separation is found in the compact crystal structure, which has an occupancy of 4-6%. Thus, CaM/4Ca2+ samples high energy local minima comprising a few discrete substates of increasing compactness in a rugged energy landscape.

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

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.