Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-α mutant unmasks a new conformation of the pathway residue 731† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03460d

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


Introduction
Coupling of electron and proton transfers between donors and acceptors in proteins are ubiquitous in biology and can occur in a stepwise or concerted fashion. The concerted case avoids high energy intermediates and is designated as proton coupled electron transfer (PCET). 1 The mechanisms of these couplings are fundamental to our understanding of photosynthesis, respiration, synthesis of DNA building blocks, and many other processes. Unresolved issues describing these mechanisms have been articulated in several recent comprehensive reviews, with different mechanisms dictated by transfer distances, protein environment and dynamics. 2,3 When the proton and electron donor and acceptor are distinct, the mechanism involves orthogonal PCET; when the donor and acceptor are the same, it involves collinear PCET. 4,5 A different mechanism in which a proton is transferred through water chains over long distances in concert with electron transfer (ET) has also been recently studied and discussed extensively in model systems. 6,7 In all mechanistic cases, since the electrons and protons have very different masses, electrons tunnel over large distances (10-15Å) while proton tunnelling is restricted to shorter distances, on the order of hydrogen bond lengths. 1,8,9 This distance dependence complicates the issue of proton management. One important representative of the diversity of PCET mechanisms in proteins is found in the class I ribonucleotide reductases (RNRs). These enzymes catalyze the conversion of nucleotides to deoxynucleotides, the monomeric precursors required for DNA replication and repair in all eukaryotic and some prokaryotic organisms. 10,11 In this paper, we use the Escherichia coli (E. coli) class Ia RNR as a model system to interrogate the PCET process across the interface of the two subunits of this enzyme, proposed to involve two redox active protein tyrosine residues, one on each subunit, and a water interface between the subunits. 12 The E. coli RNR consists of two homodimeric subunits, a2 and b2. 13 The enzyme is active when a transient a2b2 complex is formed. 14 a2 contains the active site for nucleotide reduction and two allosteric effector binding sites that regulate the spec-icity and the rate of reduction. [15][16][17][18][19] b2 harbors the essential diiron tyrosyl radical cofactor (Fe III 2 -Y 122 c). 20,21 During each turnover, Y 122 c-b2 oxidizes C 439 -a2 to a thiyl radical, which subsequently initiates dNDP production. 11 There are X-ray structures of the individual subunits, and a docking model of the a2b2 complex places Y 122 c at a distance of about 35Å from C 439 . 22,23 These initial studies led to the rst formulation of radical transfer (RT) in RNR via a radical hopping mechanism involving a pathway of conserved amino acids (Y 122 4 [W 48 ?] 4 Y 356 in b2 to Y 731 4 Y 730 4 C 439 in a2). Biochemical 24 and biophysical (EPR, 25,26 SAXS, 27 and cryoEM 28 ) studies conrmed that the docking model provides a reasonable representation of E. coli RNR in its transient, active form and led to a detailed mechanism of RT over such a long distance. 4,14,24 Nevertheless, in wildtype (wt) E. coli RNR, the rate limiting step, conformational change(s) upon substrate and allosteric effector binding to a2, has prevented spectroscopic detection of any intermediates in this process. 29 The recent development of methods to site-specically incorporate tyrosine analogs with altered pK a s and reduction potentials has permitted the detection of pathway radical intermediates [30][31][32] and, combined with state-of-the-art EPR spectroscopy, 12,26,33,34 has started to reveal the molecular basis of the long-range RT in RNR. 14 These experiments have led to the current model illustrated in Fig. 1, which involves orthogonal PCET 35 steps within subunit b2 and collinear PCET steps within the a2 subunit. 12,33 However, the mechanism of the PCET process at the subunit interface between Y 356 in b2 and Y 731 in a2 remains elusive, as structural information on the C-terminal 35 amino acids of b2, including a putative proton acceptor E 350 and Y 356 (Fig. 1), is missing. 22 Our recent high-eld (HF) EPR/ENDOR and DFT investigations using the 3-aminotyrosine mutants NH 2 Y 730 -a2 and NH 2 Y 731 -a2, which generate the corresponding NH 2 Yc upon incubation with b2, CDP (substrate) and ATP (allosteric effector), established that an unusual stacked conformation of residues 730 and 731, observed in some X-ray structures of a2 (ref. 23 and 36) (see ESI, Fig. S1 †), occurs in the a2b2 complex. 12,33 However, the X-ray structure of NH 2 Y 730 -a2 (PDB 2XO4) alone exhibited multiple conformations for Y 731 -a2, with one rotated away from NH 2 Y 730 -a2 toward the a2b2 subunit interface. 30 This "ipped" conformation was accompanied by reorientations of R 411 and N 733 in a2. Further comparison of NH 2 Y 730 c-a2, NH 2 Y 731 c-a2 and NH 2 Y 356 c-b2 by HF EPR indicated that the electrostatic environment of all three transient NH 2 Ycs is strongly perturbed and that their hydrogen bond interactions are intrinsically different. 12,33 Interestingly, one of our DFT models of the protein environment for NH 2 Y 731 c-a2 required R 411 -a2 to explain the perturbed g x value observed and suggested that R 411 -a2 approaches to NH 2 Y 731 c-a2 within 2.6Å (Fig. S1 †). 12 Therefore, to examine the role of R 411 -a2 during the PCET process in E. coli RNR, we generated two mutants: R 411 A-a2 and the double mutant NH 2 Y 731 /R 411 A-a2. Here, we report the incubation of NH 2 Y 731 /R 411 A-a2 with b2/CDP and ATP, which generates the NH 2 Y 731 c/R 411 A-a2b2 complex. Using advanced EPR methods, including 263 GHz pulse EPR and 34 GHz PELDOR/DEER (pulsed electron-electron double resonance) and ENDOR (electron-nuclear double resonance) spectroscopies, we have provided evidence for a new conformation of NH 2 Y 731 c/R 411 that is "ipped" towards the subunit interface in the a2b2 complex. This is the rst time an alternative conformation of any pathway tyrosine (NH 2 Y 731 c) has been observed and it provides a new probe of the PCET mechanism across the subunit interface, which remains unknown.
Site-directed mutagenesis to generate R 411 A-a2 and NH 2 Y 731 / R 411 A-a2 The Quikchange kit (Stratagene) was used to generate each mutant according to the manufacturer's protocol. The templates pET28a-nrdA and pET28a-nrdA Y 731 Z 30 were ampli-ed with primer 5 0 -G CAG GAA CGT GCG TCT ACC GGT GCG ATC TAT ATT CAG AAC GTT GAC-3 0 and its reverse complement and used to insert a GCG (Ala) at position 411. The sequences were conrmed by QuintaraBio Laboratory. All constructs contain an N-terminal (His) 6 -tag with a 10 amino acid linker. 30 Expression, purication and activity assays of R 411 A-a2 and NH 2 Y 731 /R 411 A-a2 (His) 6 -wt-a2 (2750 nmol min À1 mg À1 ) and wt-b2 (7000 nmol min À1 mg À1 ), and 1.2 Yc/b2 were expressed and puried by standard protocols. 30,37,38 All a2 mutants were pre-reduced with 30 mM DTT and 15 mM HU before use. 29 E. coli thioredoxin (TR, 40 U mg À1 ) and thioredoxin reductase (TRR, 1800 U mg À1 ) used in assays were isolated as previously described. 39,40 (His) 6 -NH 2 Y 731 -a2 was puried as previously described. 30 Expression and purication of R 411 A-a2 and NH 2 Y 731 /R 411 A-a2 followed previous protocols, 30 except that the purication buffer (50 mM Tris, 5% glycerol, 1 mM PMSF, pH 7.6) for NH 2 Y 731 /R 411 A-a2 contained 1 mM TCEP. The yields of puried R 411 A-a2 and NH 2 Y 731 /R 411 A-a2 were 10-12 mg g À1 and 6-7 mg g À1 cell paste, respectively. The activity of R 411 A-a2 (0. Samples for HF EPR and PELDOR spectroscopy NH 2 Y 731 /R 411 A-a2 and wt-b2 were mixed 1 : 1 to a nal concentration of 160-180 mM in D 2 O assay buffer as previously described. 32,34 These protein concentrations resulted in >95% binding between subunits. The reaction was initiated at room temperature by adding CDP and ATP to nal concentrations of 1 and 3 mM, respectively. The reactions were manually freezequenched in liquid N 2 within 10-23 s. The PELDOR sample was prepared by adding glycerol-(OD) 3 to a nal concentration of 10% (v/v) 16 s aer the initiation of the reaction. This reaction was manually freeze-quenched aer 56 s as just described. The NH 2 Y 731 c accounted for 30-33% of the total spin for all the samples used in this work, which was similar to the yields reported previously. 30,32 HF pulsed EPR spectroscopy Echo-detected (ESE: p/2 -s -pecho) EPR spectra at 263 GHz were recorded on a Bruker Elexsys E780 quasi optical spectrometer using a single mode (TE 011 ) cylindrical resonator (E9501610 -Bruker BioSpin) with a typical quality factor of 500-1000. The maximum microwave power coupled to the resonator was about 15 mW. Samples for 263 GHz EPR were inserted in capillaries (0.33 mm OD, Vitrocom CV2033S) with typical volumes of ca. 50 nL. 94 GHz ESE spectra were recorded on a Bruker E680 spectrometer with a 400 mW W-band power setup (Bruker power upgrade -2). Samples for 94 GHz ESE contained typical volumes of 2 mL in 0.84 mm OD capillaries (Wilmad S6X84). All manually freeze-quenched samples were immersed in liquid N 2 and loaded into pre-cooled EPR cryostats.

GHz PELDOR spectroscopy
34 GHz ESE and PELDOR spectra were recorded on a Bruker E580 X/Q-band spectrometer equipped with a Bruker EN 5107D2 pulse EPR/ENDOR resonator. The spectrometer was power-upgraded with a Q-band TWT amplier, providing about 170 W output power at 34.1 GHz. PELDOR experiments were recorded with an overcoupled resonator. The center of the mode was chosen for the pump frequency for measurements at 20 K. However, for measurements at 50 K the detection frequency was set in the center of the cavity mode to enhance detection sensitivity. Q-band samples contained typical volumes of 10 mL in 1.6 mm OD capillaries (Wilmad 222T-RB).

Processing and simulation of EPR spectra
Spectra were processed by phasing and baseline correction. Derivatives of the absorption spectra were obtained by tting every four points with a second order polynomial and differentiating the function in MATLAB_R2014b. 43 EPR spectra were simulated using the EasySpin-4.5.5 "pepper"-routine which was run in MATLAB. 44

DFT calculations
DFT calculations were performed with the ORCA 3.0.0 program package. 45 The geometry optimization of the neutral NH 2 Yc was performed using the unrestricted B3LYP 46-48 hybrid density functional in combination with the def2-TZVPP basis set and def2-TZVPP/JK auxiliary basis set. 49,50 To take into account the electrostatic environment of the radical intermediate at the protein interface, a solvation model (COSMO 51 ) with the polarity of ethanol (3 ¼ 24) was used. Otherwise, Grimme's dispersion correction 52,53 and RIJCOSX 54 approximations were employed. The energy converged to 10 À9 E h . The hyperne couplings and g values were calculated using NH 2 Yc-C 4 as the gauge origin. 55,56 The def2-TZVPP basis set was consistent with the geometry optimization step. 50 The C2-C1-Cb-Ca dihedral angle of the NH 2 Yc was changed stepwise with a geometry optimization for each step. The xyz coordinates for one of the optimized models are given in the ESI. †

PyMOL models
The docking model refers to the a2b2 complex structure generated from the individual wt-a2 and wt-b2 X-ray structures. 22,23 In order to predict distances, the mutant E. coli RNR structure (PDB 2XO4) 30 was overlaid with the wt-a2 structure in the docking model 23 using PyMOL, which rst performs a sequence alignment and then aligns the structures to minimize the root mean square deviation between the structures.

Results and discussion
Preparation and characterization of R 411 A-a2, NH 2 Y 731 /R 411 A-a2 and ND 2 Y 731 c/R 411 A-a Our recent studies on NH 2 Y 731 -a2 (ref. 12) suggested that R 411 might interact with NH 2 Y 731 c, partially accounting for the measured EPR and ENDOR parameters. To investigate this proposal, R 411 A-a2 was generated and characterized. Because the mutation is proposed to be at the interface of a2 and b2, the dissociation constant (K d ) for subunit interactions was also examined and was determined to be 0.94 AE 0.33 mM (Fig. S2A †), $5 fold higher than that for wt-a2 (0.18 mM). 42 Under these conditions, this mutant was shown to have a specic activity of 467 AE 22 nmol min À1 mg À1 , 17% of that of the wt enzyme (2750 nmol min À1 mg À1 ). The reduced activity and weaker subunit binding suggest that R 411 plays a functional role.
Furthermore, we characterized the role of R 411 in the oxidation of Y 731 -a2 by generating the double mutant NH 2 Y 731 /R 411 A-a2. The K d for subunit interactions between NH 2 Y 731 /R 411 A-a2 and wt-b2 was determined to be 8 AE 1 nM (Fig. S2C †), which is consistent with the formation of a tight complex when a NH 2 Yc is generated. 28 Its specic activity was 13 AE 3 nmol min À1 mg À1 , 0.4% of the specic activity of wt-RNR and in the range of contaminating wt-a2 activity. 32 A more sensitive, one turnover assay was then employed to determine if this double mutant could generate any dCDP. When pre-reduced NH 2 Y 731 /R 411 A-a2 was mixed with wt-b2, CDP, and ATP for 5 min, only 0.036 AE 0.018 dCDP/a2 was observed, consistent with contaminating wt-a2. Thus, the double mutant is unable to make detectable dCDP, which is not unexpected, given the specic activities of the R 411 A and the NH 2 Y 731 -a2 mutants (see also SI-3 and Fig. S3 †).
We next investigated whether NH 2 Y 731 c could be generated by NH 2 Y 731 /R 411 -a2, despite its inability to make dCDPs. NH 2 Y 731 /R 411 A-a2, wt-b2, CDP and ATP were studied by stopped-ow (SF) spectroscopy and the reaction was monitored at 320 nm, the absorption feature associated with the NH 2 Yc (Fig. S4, † red). The data were split into two time domains: 5 ms to 6 s and 25 s to 100 s. In the rst time domain, NH 2 Y 731 c formation was t to a double exponential with k fast of 3.6 AE 0.5 s À1 (amplitude 8%) and k slow of 0.47 AE 0.03 s À1 (amplitude 21%) (Table S1 †). The rate constants for NH 2 Y 731 c in the single mutant control were similar: k fast of 9.6 AE 0.6 s À1 and k slow of 0.8 AE 0.1 s À1 . However, in this case, the fast phase accounted for 27% and the slow phase accounted for 13% of the NH 2 Y 731 c. The biphasic kinetics of NH 2 Y 731 c formation in both cases is attributed to multiple conformations that give rise to NH 2 Y 731 c. 32 From 25 s to 100 s, NH 2 Y 731 c in the double mutant reaction disappeared with a k obs of 0.02 AE 0.003 s À1 , while with the single mutant, disappearance occurred with a k obs of 0.005 AE 0.002 s À1 . Analysis of the Y 122 c-b disappearance kinetics was unsuccessful at early time points due to the detection limits, as described in SI-4.
Given the distinct kinetics of our double mutant relative to the NH 2 Y 731 -a2, the 9 GHz EPR spectrum of the sample generated from the reaction of NH 2 Y 731 /R 411 A-a2 with wt-b2, ATP, and CDP quenched aer 25 s was recorded and is shown in Fig. S5A and C. † Subsequent to subtraction of Y 122 c, 32% of the total spin is associated with NH 2 Y 731 c/R 411 A-a2 with no spin loss. This result is similar to that of the single mutant, NH 2 Y 731 c. 30,32 A comparison of their spectra, as shown in Fig. S5B, † revealed substantial differences in their hyperne interactions, suggesting that further characterization of this radical might provide insight into the function of R 411 . Therefore, the role of R 411 in the RT pathway was further studied with advanced EPR spectroscopy.
HF EPR of ND 2 Y 731 c/R 411 A-a2 To examine the generated ND 2 Y 731 c/R 411 A-a2, we took advantage of the proximity of Y 122 c to the di-iron cluster and its altered relaxation properties. Pulsed EPR spectra of ND 2 Y 731 c/R 411 A-a2 at 34, 94 and 263 GHz were recorded in D 2 O buffer at 70 K and are shown in Fig. 2A. The use of D 2 O considerably simplies the EPR spectra due to the absence of 1 H hyperne (hf) splittings arising from the amino protons. The ND 2 Y 731 c/R 411 A-a2 EPR spectrum at 34 GHz is mainly dominated by the large hf couplings with the deuterons of the amino group and the two Cb-methylene protons. 34 On the other hand, the 94 and 263 GHz EPR spectra are dominated by g-anisotropy, and the relative contributions of g-and hf-anisotropy are strongly dependent on the operating magnetic eld. The g values of ND 2 Y 731 c/R 411 A-a2 are best resolved at 263 GHz and are consistent with the values from our previous ND 2 Yc studies. 12,33 The 94 GHz spectra reveal differences in the hf splitting of the Cb-methylene protons ( Fig. 2A, marked with an arrow): the large hf splitting of the Cbmethylene proton visible in the central line of ND 2 Y 731 c-a2 (red) is missing in ND 2 Y 731 c/R 411 A-a2 (black). This splitting is also absent in the 263 GHz spectrum. The EPR spectra were simulated iteratively to nd a global solution for the contributing hf couplings. All of the EPR data and simulations, in which the previously reported 34 hf coupling for 14 N is used, are consistent with the NH 2 Y 731 c generated in the NH 2 Y 731 c/R 411 A-a2/b2 complex being a single, well-oriented radical species with one set of magnetic parameters, which are listed in Table 1 (see also Fig. S7 †). This nding is not self-evident, as our previous experiments with other double mutants, NH 2 Y 731 c/Y 730 F-a2 and NH 2 Y 730 c/C 439 A-a2, showed distributions in g values indicative of multiple radical environments and/or molecular orientations. 12 Interestingly, we do not observe changes in the g values between ND 2 Y 731 c/R 411 A-a and ND 2 Y 731 c-a2. This is unexpected because the g x value is affected by the electrostatic environment of a radical, 57 and the R 411 A mutation has changed the local environment of ND 2 Y 731 c, as demonstrated by the substantial changes in the Cb-methylene 1 H couplings (Table 1). These couplings are related to the dihedral angle q Cb between the Cb-H bond and the p z orbital axis of C 1 (Fig. 2B), and therefore provide information on the molecular orientation of the tyrosyl and 3-aminotyrosyl radicals. 34 The dihedral angle can be extracted from the McConnell equation (a iso(Cb-H) ¼ B 1 Â r C1 Â cos 2 q Cb ), 58 which provides a semi-empirical relationship for the observed isotropic constant a iso . The C2-C1-Cb-Ca angle of ND 2 Y 731 c/R 411 A-a2 is estimated to be z90 by using B 1 of 162 MHz (ref. 59) for tyrosyl radicals, an electron spin density r C1 of 0.214, 12 and an isotropic Cb-methylene proton hf coupling a iso ¼ 10 AE 1 MHz (Table 1). This dihedral angle is indeed consistent with the hf couplings of the two Cb-methylene 1 H resonances being indistinguishable, as reported in Table 1 and seen in Fig. 2B and C. This result was conrmed by DFT calculations on the observed hf couplings of NH 2 Yc, in which the ring orientation was modeled with respect to the backbone and showed a symmetric orientation relative to the p z orbital axis of C 1 (Fig. 2B). In this calculation, a q Cb angle of 90 corresponds to a iso ¼ 9 AE 3 MHz (grey area in Fig. 2C) for both Cb-methylene protons, H b2/1 .

ENDOR for detection of hydrogen bonds to ND 2 Y 731 c/R 411 A-a2
Given that the R 411 A mutation had little effect on g x , 2 H ENDOR spectroscopy was used to further examine a possible correlation of the observed g x value (g x ¼ 2.0051) with the hydrogen bonding environment. Fig. 3 illustrates the 2 H Mims ENDOR spectra of ND 2 Y 731 c-a2 and ND 2 Y 731 c/R 411 A-a2. Both spectra contain a broad signal that extends over AE2 MHz, arising from the strongly coupled amino deuterons, which is a common feature of ND 2 Yc Mims ENDOR spectra. 12,33 However, we observe that the 2 H hf tensor previously assigned to the moderately strong hydrogen bond between Y 730 and Y 731 in ND 2 Y 731 c-a2, which is almost perpendicular to the tyrosine ring plane, 12 is absent in the ND 2 Y 731 c/R 411 A-a2 spectrum. Therefore, the hydrogen bonding environment of NH 2 Y 731 c/R 411 A-a2 is distinct from that of the single mutant, consistent with the different side chain conformations observed by HF EPR spectroscopy. Note that almost the complete EPR line of ND 2 Y 731 c/R 411 A-a2 can be excited at 34 GHz by using very short microwave pulses,  and thus hf couplings cannot be missed due to orientation selective effects. Although no exchangeable moderately strong hydrogen bonds (r O-H $ 1.7-2Å) to ND 2 Y 731 c/R 411 A-a2 are observed, the ENDOR spectrum of ND 2 Y 731 c/R 411 A-a2 exhibits a broad and structured matrix line, which is associated with weak hf interactions of the radical with distant nuclei 61 (see Fig. 3, inset). The structure in this matrix line suggests the presence of weakly coupled deuterons that cannot be resolved from the matrix ones (matrix line). We note that the ENDOR spectrum of ND 2 Y 731 c/ R 411 A-a2 is reminiscent of the one previously observed for ND 2 Y 356 c-b2, also located at the subunit interface and likely surrounded by a dened hydrogen bonded network of water molecules. 12 The similarity between the ENDOR spectra of ND 2 Y 356 c-b2 and ND 2 Y 731 c/R 411 A-a2 suggests a similar origin for the g x values in these two mutants, which is distinct from that in ND 2 Y 731 c-a2. As noted above, in the case of ND 2 Y 356 c-b2 the g x value was also strongly shied (NH 2 Y 356 c: g x ¼ 2.0049 vs. free NH 2 Yc: g x ¼ 2.0061 (ref. 33)). Therefore, we propose that the g x -shi in NH 2 Y 731 c/R 411 A-a2, as well as in ND 2 Y 356 c-b2, arises from weakly coupled hydrogen bonds observed in the 0.3 MHz region of the ENDOR spectrum. The complexity of the g tensor interpretation was underlined by our recent DFT calculations, in which three distinct models for NH 2 Y 731 c-a2 resulted in similar g-shis. 12 Overall, these data clearly indicate that the molecular orientation of ND 2 Y 731 c/R 411 A-a2 is different to that of ND 2 Y 731 c-a2 and is affected by R 411 A-a2 substitution.
PELDOR gives evidence for a conformational change in ND 2 Y 731 c/R 411 A-a2 Our previous PELDOR studies 26 have demonstrated that half sites reactivity of E. coli RNR allows for the detection of the diagonal inter-spin distance between Y 122 c in one ab pair and any radical trapped in the second ab pair (Fig. 4A). 25,62 To gain insight into the location of NH 2 Y 731 c/R 411 A-a2, three sets of PELDOR experiments were recorded using broadband excitation with a high-power Q-band set up at different excitation positions in the EPR line 63-66 (see Fig. 4B and S8 †). The recorded time traces are displayed in Fig. 4C and show substantial differences in modulation depth (10 to 50%), which is typical for orientation selection effects. Trace D1 also shows a higher frequency component that arises from the parallel component of a dipolar Pake pattern (Fig. S8 †). For this reason, the background corrected PELDOR time traces from the three sets of experiments were summed and the resulting trace was analyzed as shown in Fig. 4C and D. Additional comparison of the Fourier-transformed traces (Fig. S8 †) shows that the sum trace leads to an almost complete Pake pattern. Distance distribution analysis revealed a clear dominant peak at 35Å with a distance distribution of Dr ¼ AE2.7Å. We note that the error in the peak distance is much less than the distribution and is estimated to be # AE0.5Å. The width of the distance distribution is slightly larger than in previous measurements within the E. coli RNR a2b2 complex, 25,26,62 suggesting more conformational heterogeneity for ND 2 Y 731 c/R 411 A-a2, consistent with the observed exibility of this residue. Nevertheless, the results clearly indicate that the R 411 mutation induces a conformational change of ND 2 Y 731 c into a new well-dened conformation.
The peak distance of 35.0Å has never been observed between any radicals formed in this pathway before, and it is 3Å shorter than that previously measured for ND 2 Y 731 c-a2. 26 This distance might appear to be rather close to the initial distance (prior to turnover) between the two stable Y 122 cs, that is 33.1 AE 0.2Å. 62 To conrm our assignment, we recorded PELDOR experiments at higher temperature (50 K), in which the Y 122 c-b2 contribution to the re-focused echo is ltered and ND 2 Y 731 c-a2 is the only radical species detected (Fig. S9 †). However, Y 122 c-b2 can still be excited by the pump pulse and contributes to the PELDOR signal. Under these conditions, any distance observed in the PELDOR experiments at 50 K is related to Y 122 c-ND 2 Y 731 c and cannot be associated with the Y 122 c-Y 122 c distance, as the latter radical is not detected. The distance distribution analysis of the 50 K measurements yielded a peak distance of 35.3Å with a distribution of Dr ¼ AE 2.0Å, and thus validated our assignment (see Fig. S9 †).
To gain more insight into the conformation of NH 2 Y 731 c/ R 411 A-a2 and the role of R 411 , we examined the available X-ray structures of E. coli a2s in the R 411 region. In the structure of E. coli NH 2 Y 730 -a2 (2XO4), 30 Y 731 is ipped away from NH 2 Y 730 , as shown in Fig. 5. This altered conformation is compared with a second a in the unit cell, in which the Y 731 is not ipped. To match the 35Å distance observed by PELDOR spectroscopy, the aromatic ring of NH 2 Y 731 must rotate away from Y 730 toward the b2 subunit, as observed for Y 731 in the E. coli Y 730 NH 2 Y-a2 structure (Fig. 5). This reorientation is also supported by the ENDOR data, which indicate that the stacked conformation between NH 2 Y 731 c and Y 730 with a shared, perpendicular hydrogen bond is absent in NH 2 Y 731 c/R 411 A-a2, and that the radical is instead surrounded by weakly coupled hydrogen bonds, likely water molecules at the a2b2 subunit interface. The exposure of NH 2 Y 731 c/R 411 A-a2 to the interface and the buffer in this new conformation might be the origin of the instability of the radical as compared to the single mutant (Table S1 †).
We have also examined another possible conformation, in which the amino group of NH 2 Y 731 -a2 moves to occupy the vacancy created by the mutation of arginine to alanine. This conformation is displayed in Fig. S10. † However, in this case the expected distance between the oxygen atoms of NH 2 Y 731 and Y 122 exceeds the observed distance by $2Å. We note that the "ipped" conformation has not been observed in the single mutant NH 2 Y 731 -a2 or in the double mutant NH 2 Y 731 /Y 730 F-a2, in which Y 731 lacks its hydrogen bonding partner, 12 suggesting the importance of R 411 in stabilizing the stacked conformation. This change between a ipped and non-ipped conformation of the interface Y might play an active role in the PCET process between Y 731 and Y 356 in wt RNR, the mechanism of which is still not understood. With the wt enzyme, this conformational change is kinetically masked by physical gating, which ratelimits RNR, and is too fast to be detected based on the recently   5 The E. coli Y 730 NH 2 Y-a2 structure (2XO4) 30 in green shows the reoriented Y 731 overlaid with the stacked Y 731 in a different monomer (blue) of the unit cell. The diagonal distances between the "flipped" and non-flipped Y 731 and Y 122 are 3.5 nm and 3.9 nm, respectively. These distances, which are between two phenolic oxygen atoms of the tyrosine residues, are based on the alignment with the E. coli a2b2 docking model. Residue Y 356 is shown in grey with a "?" because its position is unknown. measured rate constants for electron transfer (ET) (10 4 to 10 5 s À1 ) at the interface by photo-RNRs that unmask this gating. 68,69 Thus, the R 411 A mutation might have fortuitously allowed detection of this movement at the subunit interface.
While the lack of structural information at the subunit interface poses a challenge for a mechanistic understanding of interfacial PCET, the detection of the NH 2 Y 731 c/R 411 provides us with a spectroscopic probe of this interface. Mutagenesis and site-specic isotopic labeling of interface residues could provide us with additional insight into how this step is controlled. Finally, the mechanism of PCET across the subunit interface observed with the E. coli RNR is likely to be conserved in all class I RNRs based on their subunit structures and the conserved weak subunit associations dictated by the C-terminal tail of b2. 70,71 The pathway for oxidation is conserved between RNR classes Ia, Ib and Ic, as is the regulation of the pathway by NDP/ effector binding. 72 Thus, while the "details" of the radical transfer mechanism might be different in the individual class I RNRs, general principles will likely emerge from the studies on E. coli RNR, given all of the evolutionarily conserved features.

Conclusions
This study has revealed that the E. coli RNR double mutant NH 2 Y 731 /R 411 A-a2 unmasks a new conformation of pathway residue 731 in the a2b2 complex. This is the rst experimental evidence for the exibility of this pathway or any pathway residue in the active enzyme. The results have provided insight into the mechanisms of PCET within a2, as well as through the a2b2 interface. First, R 411 appears to play a role in the stabilization of the stacked conformation of Y 731 and Y 730 , and thus in the facilitation of collinear PCET within the a2 subunit. Second, the new conformation is consistent with Y 731 pointing toward the subunit interface, in the direction of the adjacent pathway residue Y 356 , located in the exible C-terminal tail of subunit b2. The exibility of these two contiguous pathway residues, which have been suggested to communicate during PCET, 69 might be the key to driving the RT chemistry at the subunit interface through water clusters. 6,7 This opens up a new hypothesis for the PCET mechanism between residues Y 731 -a2 and Y 356 -b2, which could involve a gated conformational change in Y 731 -a2 in wt RNR on a fast time scale, not observable without the R 411 A mutation. While this hypothesis remains to be proven, the present results will serve as a basis to design new experiments aimed at detecting a possible combined role of Y 731 -a2 and Y 356 -b2 in PCET through the subunit surface.