Protein expression and purification

B. cereus R2F, NrdI, and the thioredoxin like protein, BC3987 (and its thioredoxin reductase), were expressed and purified as previously described [24], [37], [38]. The extinction coefficient was determined using the Edelhoch method [39].

Sample preparation

After purification, the RNR R2F protein was pretreated with 10 mM hydroxyurea and 5 mM EDTA at 4°C for 20 min and passed over a 5 mL HiTrap desalting column (GE healthcare) to remove the iron and to reduce the tyrosyl radical before reconstitution with Fe^II. The iron-tyrosine radical site in B. cereus R2F was generated by addition of excess Fe^II and O₂ and incubated at 0°C for 10 minutes. The final volume of the EPR samples was 180–200 µL in 50 mM HEPES, 100 mM KCl, and 20% glycerol (v/v), pH 7.5. Samples of active B. cereus R2F for resonance Raman were prepared identically, but protein was diluted into 50 mM Tris-HCl, 100 mM KCl, and pH 7.5. Deuterated resonance Raman samples were prepared by diluting and re-concentrating apo protein in pure D₂O (99.9%D, Cambridge Isotope Laboratories) and then in 50 mM Tris-HCl, 100 mM KCl, pD = 7.9 using Amicon Ultra-15 (Millipore) 7 times. ¹⁸O samples were prepared by incubating for approximately 45 min in H₂¹⁸O after formation of the radical [40]. The in vitro generation of the dimanganese-Y^• cofactor was carried out as previously described using NrdI_hq (hydroquinone form of NrdI) in the reacting the mixture with oxygen [20]. After tyrosyl-radical formation the HF-EPR sample, was additionally passed over a 5 mL HiTrap desalting column (GE healthcare) to remove most of the Mn(II) not in di-Mn cluster and finally the sample was concentrated to about 200 µl by centrifugation on a Millipore/Amicon microcon YM-30.

UV/Vis spectrophotometric assays

Light absorption spectra were measured on a Hewlett-Packard 8452 diode array spectrophotometer in the wavelength range of 250–700 nm using 1 cm optical-path quartz cuvette.

X-band EPR experiments

EPR spectra were recorded at X-band on a Bruker Elexsys 560 EPR spectrometer fitted with a Bruker ER41116DM dual-mode cavity and using a He-flow cryostat (ESR 900, Oxford Instruments). The sample spin concentration was obtained by comparing double integrals of spectra with 1 mM Cu^II EDTA prepared in a solution of 50 mM HEPES, pH 7.5, 20% (v/v) glycerol, recorded under non-saturating conditions. All spectra were measured under identical non-saturating microwave power. First-derivative EPR spectra were recorded at different microwave power (P) and at various temperatures to determine the microwave power at half saturation (P_1/2) for each temperature examined. The data were fitted with the Equation 1 [41], [42], [43].(1)The term ∫∫S represents the double integrated signal intensity (S), P is the applied microwave power, b is the relaxation factor (b = 1 for inhomogeneous line broadening and b = 3 for homogeneous line broadening) [41], [42], P_1/2 is the power at which the signal is half-saturated and k is an experimental constant associated with the instrument. In order to apply Eq. 1 the following experimental conditions must be satisfied: i) the samples should be in a region of the cavity with maximum microwave field (H₁), thus the filling factor needs optimization, ii) the sample temperature must be constant and iii) the frequency and the gain must also be constant. Eq. 1, however, is not strictly applicable when dipolar couplings and/or exchange interactions are effective in the system. This is due to the fact that the half-saturation power (P_½) is defined by Eq. 2 and Eq. 3 where V represents the cavity volume, Q the cavity quality factor (Q = H₁²V/2P), P the power dissipated in the cavity and γ the gyromagnetic ratio. Eq. 2 assumes that all spins at resonance saturate equivalently, hence they feature the same product (T₁T₂).(2)(3)When dipolar and/or exchange interactions are present in the spin system, the product (T₁T₂) is not constant thus 1≤b≤3 is no longer valid and b becomes less than 1 [44], [45], [46]. The simulated EPR spectra were computed with two programs. (i) The Bruker Biospin XSophe V.1.1.4 (matrix diagonalization, g-strain, where the strain is defined as Δg = ge µB/h×(ΔB/ν), with strain imposed Δg_(x,y,z) = 0.0001). (ii) The simulation platform SimFonia (Bruker biospin, V. 1.25), which uses powder perturbation theory but without additional contributions to signal broadening due to g-strain effects. We observed that with X-Sophe (g-strain model), we could simulate by using the same line-width tensor both X-band and High-Field EPR spectra. The use of the same line-width tensor as employed in the X-Sophe platform by SimFonia did not allow to reproduce satisfactorily the observed X-band spectrum of Fe B. cereus RNR R2, and only a much larger line-width tensor (L_x,y,z as shown in Table 1) gave comparable well reproduced spectrum (see Figure S1). We did not use the software SimFonia for simulation of the High-Field EPR spectrum of Fe loaded RNR R2F form. Estimation of the θ angle, namely the angle between tyrosyl plane versus plane generated by the ethyl residue (Figure 1B) can be obtained following two approaches; (1) Through simulation under spin-Hamiltonian approximation the best set of anisotropic proton hyperfine couplings (H-hfc) (A) terms for the H_β1 and H_β2 protons are obtained, which can be used to fed the hfc H_β1, H_β2 (Y) vs θ (X) diagram derived by theory by Himo and Gräslund [47], or (2) by using the empirical relation developed by Svistunenko and Cooper [48] where only two input variables θ and ρ_C1 (McConnell spin density) can be used to simulate the entire EPR envelope; for example, for a fixed combination of θ- and ρ_C1-values, A_(β1)iso and A_(β2)iso can be extracted and compared with those determined experimentally. In this work, for estimation of θ we did use the first approach (1). Sign and magnitude of hfc terms for H_β1, H_β2, H3 and H5 were initially derived from DFT analyses H_β1, H_β2 hfc values were then compared to those computed by Himo and Gräslund [47], followed by stepwise deconstruction of the EPR resonance line, so to refine the anisotropic H-hfc components for H3 and H5 protons that generated a simulated EPR envelope consistent with the recorded experimental spectra.

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Table 1. The g–tensors, hfc–tensors (A, mT), linewidths (LW, mT), and rotational θ angle (°) for the tyrosyl radical in class Ia and class Ib RNRs and the Y_D radical in photosystem II, *Inequivalent C₃ and C₅ protons, **Using XSophe (v. 1.1.4) (matrix diagonalization, g-strain model).

https://doi.org/10.1371/journal.pone.0033436.t001

High-field EPR measurements

The low temperature 285 GHz (high-field/high-frequency EPR, HF-EPR) spectra were obtained with a 95 GHz Gunn oscillator (Radiometer Physics, Germany) coupled to a frequency tripler as the frequency source and a superconducting magnet with a maximum field of 12 T at 4.2 K (Cryogenics Consultant, UK) for the main magnetic field. Temperature of sample was changed by a variable temperature Insert (Oxford Instruments, UK) over a temperature range from 1.5 K to 300 K, with the sample directly in the helium flux. The detection of the light transmitted through the sample was performed with a ‘hot electron’ InSb bolometer as described [33], [49].

Computational procedures

The theoretical modeling of the tyrosyl radical was performed in the gas phase by density functional theory (DFT) considering the radical in the uncharged form (neutral) with the spin unrestricted B3LYP functional (Exchange of 0.2000 Hartree-Fock, 0.0800 Slater and 0.7200 Becke, correlation of 0.8100 LYP and 0.1900 VWN1RPA) using the Euler-Maclaurin-Lebedev (EML grid; 70,302) quadrature formula and basis set 6-311++G(d,p) as implemented in the computational package Spartan 10. The molecular structure of the tyrosyl molecule was optimized under constrained θ (C6-C1-Cα-Cβ) torsional angle (root mean square gradient below 10⁻⁷), followed by frequency calculation to derive vibrational frequencies. Single point calculations were carried out on the optimized geometry with Gaussian 03 (Version 6.0) using the same functional but tighter SCF convergence (SCF = tight) to obtain accurate Mulliken atomic spin-population analyses. The list of vibrational frequencies (cm⁻¹) and zero point vibrational energies are provided in the Calculation S1.

Resonance Raman spectroscopy

Resonance Raman spectra were recorded using a Spex 1877 CP triple monochromator with 1200, 1800, and 2400 grooves/mm holographic gratings and an Andor Newton CCD detector cooled at −80°C. Excitation was provided by either a Coherent I90C-K Kr+ ion laser (λ_exc = 406.7 nm) or an Innova Sabre 25/7 Ar+ CW ion laser (λ_exc = 379.5 nm). The spectral resolution was ∼2 cm⁻¹. Spectra were recorded on sampled cooled in a finger Dewar with liquid nitrogen (77 K) at a power of 5 mW (379.5 nm) or 10 mW (406.7 nm) at the sample. Reported spectra are the sum of 30 to 60 accumulations of 10 to 30 seconds and 5 independent measurements using full vertical binning on the CCD. Baseline spectra were collected using ground activated charcoal, buffer and apo protein. The rRaman spectra were also collected with a Jobyn Yvon Horiba T64000 instrument equipped with a 410 nm Kaiser Optical holographic Super-Notch filter to serve as a single spectrograph to avoid high losses of Raman light. Here a three stage laser system was used as the light source: A Spectra-Physics Millennia Pro 12sJS Nd∶YAG solid state laser (6.5 W at 532 nm) pumped a Sirah Matisse TR Ti∶Sa ring Laser that produced 1 W at 820 nm. This was finally doubled to 410 nm in a Spectra-Physics Wavetrain frequency doubler (550–990 nm) to yield 20 mW of laser light with a line width of better than 4 MHz. The power of the 410 nm laser at the sample was ∼5 mW due to losses at instrument mirrors and lenses. The low band width and the small shifts of the Raman peaks investigated required an entrance slit width of 100 µm and a grating with 3600 grooves per mm to ensure sufficient resolution. 60 scans of 60 seconds were averaged for each Raman spectrum. The fluorescence signal that inevitably occurred was subtracted by fitting with a polynomial function, and the frequency scale was calibrated using 4-acetamidophenol. This resulted in a final precision that was ∼2.5 cm⁻¹ over this region. The reference peak values were obtained from literature tables [50].

Reconstitution of the tyrosyl radical in B. cereus R2F

The “apo” B. cereus R2F (Figure 2A, dark yellow line) can be readily reconstituted with excess ferrous iron and O₂ (Figure 2A, blue line). Inductively coupled plasma (ICP)-analysis demonstrated that “apo” protein contains ∼0.25 equivalents of iron per dimer subsequent to treatment with chelating agents [37]. Circular dichroism (CD) studies prove that this spurious iron bound to the protein does not form the dinuclear metal cluster and does not take part in the process of tyrosyl radical formation after oxygen binding and oxidation of the active dinuclear metal core [37]. The UV-visible spectrum of the iron loaded R2F protein (radical and oxy form, active) displays a sharp peak at 408 nm and broader bands at ∼325 and 365 nm. The former signal is characteristic of a tyrosyl radical and the latter signals of a di-iron-oxo cluster (Figure 2A, blue line). The maximum yield of tyrosyl radical was obtained with 7 equivalents of ferrous iron per R2F dimer. From EPR, the radical yield is ∼1 per dimer (see below). From UV-vis absorption the radical concentration was estimated as ∼0.8 per dimer. The discrepancy in the estimated radical content could arise from the difference in protein concentrations used in the two methods (50–75 µM for UV-vis and 200 µM for EPR). The tyrosyl radical in the active (oxy) Fe loaded protein was relatively stable and its absorption feature did not change during 30 min of incubation at room temperature. The radical signal completely vanished after 5 min when the active (oxy) Fe loaded protein was treated with 4 mM hydroxyurea (HU), which is a common radical scavenger for RNR R2 (Figure 2A, red line).

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Figure 2. UV-Vis spectra of reconstituted R2F.

(A) UV-Vis spectrum of 75 µM R2F prior to reconstitution (dark-yellow line), R2F reconstituted with ferrous iron (blue line), and after incubation with 4 mM HU for 5 min (red line) at room temperature. (B) UV-Vis spectrum of 50 µM Mn^II₂-R2F reconstituted with 25 µM of NrdI_hq and O₂ (g) (blue line), 50 µM Mn^II₂-R2F with 25 µM of NrdI_ox (dark-yellow line), and manganese reconstituted R2F treated with 4 mM HU for 5 min at room temperature (red line).

https://doi.org/10.1371/journal.pone.0033436.g002

The tyrosyl radical in B. cereus R2F can also be formed when manganese metal ions occupy the oxygen binding site, but only in the presence of NrdI, as recently shown for E. coli class Ib R2F [20], [21] and C. ammoniagenes RNR [26]. Mn^II₂-R2F was initially incubated anaerobically with the hydroquinone form of NrdI (NrdI_hq). When the sample was exposed to O₂ gas, a sharp tyrosyl radical-type absorption feature at 408 nm appeared (Figure 2B, blue line) in addition to the generation of oxidized NrdI (NrdI_ox) (Figure 2B, dark yellow line). Maximum radical formation was obtained after incubation with 4 equivalents of Mn(II) per R2F dimer, monitored at 408 nm. The radical signal decayed during incubation, both at room temperature and on ice, and about half of the radical UV-vis absorption is lost (result not shown) after 30 minutes on ice temperature (T∼4°C). When the oxidized Mn(III) cluster R2F-Tyr^• was incubated with HU for 5 min, the radical absorption signature at 408 nm disappeared (Figure 1B, red line). Control experiments show that the 408 nm does not originate from NrdI_ox, which is included in the reconstitution (Figure 2, dark-yellow line). These results clearly indicate that R2F tyrosyl-radical can be generated successfully with NrdI_hq. Finally, the manganese loaded R2F-Tyr^• protein is less stable then the iron loaded R2F-Tyr^• form, both at room and at ice temperatures.

Low temperature electron paramagnetic resonance studies of B. cereus R2F tyrosyl-radicals

Formation of a tyrosyl radical occurs together with metal-cluster oxidation. The electronic/magnetic fingerprints of the formed tyrosyl radicals were assessed with low-temperature EPR spectroscopy. Differences in the radical resonance envelope with different Fe^III₂-R2F-Tyr^• or Mn^III₂-R2F-Tyr^• cofactors were observed. The tyrosyl radical in R2F-Fe^III₂-Tyr^• was measured over a temperature range 4–77 K with two EPR microwave frequencies 9.6 (X-band) and 285 GHz (HF-EPR). The spectra are shown in Figure 3 (X-band, T = 20 K, lower black line, Obs) and Figure 4 (HF-EPR, T = 5 K, upper line, Obs) together with simulations. The anisotropic g-tensor components associated with the tyrosyl radical are poorly resolved at X-band frequency (Figure 3), however, they are clearer at high-frequency (Figure 4, T = 5 K) with g₁ = 2.0090, g₂ = 2.0044, and g₃ = 2.0021. The X-band EPR spectrum from R2F-Fe^III₂-Tyr^• of B. cereus exhibits similar resonance signature to that observed for the close homologue B. anthracis R2F [35]. Both have an EPR spectral width of ∼6.0 mT. Similar to B. anthracis R2F, the EPR signal in B. cereus R2F displays several well resolved anisotropic hyperfine splitting components (hfc) from the magnetic interaction of the unpaired tyrosyl radical spin with magnetically non-equivalent hydrogen nuclei of the tyrosine backbone. The 285 GHz spectrum does not show resolved hfc even using 0.4 mT modulation amplitude. Poor resolution of the hfc at high-fields has been observed previously in the HF-EPR spectrum of mouse R2, which contains even stronger H-hfc terms [51], [52]. It has been well established, both theoretically and experimentally, that the rotational configuration of the tyrosyl ring, namely the dihedral angle (θ) (Figures 1B and 5, see earlier description in materials and methods), strongly influences the values of the H-hfc in the tyrosyl backbone, which also depends on spin-density distribution of the tyrosyl radical specially on C1 [53]. Thus, in view of the similarities observed in the EPR envelope with B. anthracis R2F, the dihedral angle (θ) of the tyrosyl radical in B. cereus R2F was estimated as ∼60° [47], [48]. This conformation accounts well for nearly identical spin-density (from Mulliken population analysis) located on the H3,H5 protons, and for the large difference in spin-density between the β_1,2-protons (Figure 5). The derived EPR parameters are collected in Table 1 together with g, A and θ values for the tyrosyl radicals in RNR R2 from E. coli R2 [52], [54], mouse R2 [34], [49], [51], [52], [55], B. anthracis R2F [35], S. typhimurium R2F [33], M. tuberculosis R2F [56] and photosystem II (PS II) Y_D [33], [57]. The estimated angle (θ) in B. cereus Fe^III₂-R2F differs significant from those observed in the other class Ib (θ = 75°) and class Ia (θ = 30–35°) RNRs (see Table 1) [33], [35], [47], [48], [55], [56]. However, the g₁-value observed in B. cereus R2F is similar to those found in class Ib R2F proteins and class I E. coli R2 [33], [54], [56], [58]. Observation of high g₁-value suggests that the radical moiety is not involved in a hydrogen bonding interaction with nearby groups such as water molecules. For further discussion of hydrogen bond effects, on tyrosyl-radicals g₁-value and changes in spin densities between C4 and O1(Figure 1B), we refer to the following references [48], [52], [54], [55], [59], [60]. The spin concentration (S_c) of the tyrosyl radical was ∼1 spin per dimer. Moreover, S_c remained nearly constant throughout the temperature range examined (T = 4–77 K).

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Figure 3. X-band (9.66 GHz) EPR spectrum of the R2F-Fe^III₂-Tyr^• from B. cereus R2F (200 µM) recorded at T = 20 K, 12 µW microwave power, 100 KHz modulation frequency, 0.2 mT modulation amplitude and 2 scans (Obs).

The spectrum simulation (traces olive, purple, blue and red) show the EPR resonance line where only the anistropic g-tensor components are considered (olive line), when contribution from anisotropic hyperfine terms (A) due to the two H3, H5 protons (purple line, +A-H3, H5) are included, by adding further contribution arising from the Hβ₂ proton (blue line, +A-H3, H5+A-Hβ₂), and the final simulation containing all relevant contributions to the EPR envelope (red line, +A-H3, H5+A-Hβ₂+A-Hβ₁). List of simulation parameters (g-tensor, A-tensor, Line-width tensor) are given in Table 1).

https://doi.org/10.1371/journal.pone.0033436.g003

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Figure 4. HF-EPR (285 GHz) spectrum of R2F-Fe^III₂-Tyr^• from B. cereus R2F (200 µM) recorded at T = 5 K, with a modulation amplitude of 1.5 mT (Obs) and its spectrum simulation (Sim).

https://doi.org/10.1371/journal.pone.0033436.g004

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Figure 5. Spin-density distribution of an isolated tyrosyl radical obtained by density functional theory (DFT/UB3LYP/6-311++G(d,p) in gas phase, neutral form after geometry optimization (<S²> = 0.7507, the dihedral angle θ was constrained to 60°).

In this constrained conformation, the calculated atomic spin densities (from Mulliken population analyses) reveal much larger positive values located on H(β1) with respect to the H(β2) proton.

https://doi.org/10.1371/journal.pone.0033436.g005

The X-band EPR spectrum of the tyrosyl radical in R2F-Mn^III₂-Tyr^• B. cereus R2F recorded at (T = 20 K) is shown in Figure 6 (upper line, Obs) together with one simulation (lower line, Sim). The recorded resonance and derived g-tensor parameters (using spectrum simulation, g₁ = 2.0094, g₂ = 2.0039, g₃ = 2.0022), thus are only tentative but show alteration of the magnetic fingerprints of the tyrosyl radical when manganese-ions replaces two iron ions. Furthermore, the loss of resolved A_H-hyperfine resonance signals accompanied by signal broadening (∼8.5 mT) is evident in the R2F-Mn^III₂-Tyr^• spectrum. The list of R2F-Mn^III₂-Tyr^• EPR simulation parameters (values enclosed in parentheses, tentative) are given in Table 1. The dihedral angle (θ) of the tyrosyl radical in B. cereus R2F-Mn^III₂-Tyr^• was estimated as ∼65°. The spin concentration of the tyrosyl radical was determined in this case as ∼0.25 spin per dimer. The g₁ resonance feature of the B. cereus R2F-Fe^III₂-Tyr^• HF-EPR signal can also be observed in the HF-EPR spectrum of R2F-Mn^III₂-Tyr^• (Figure S2, T = 5 K). However, the entire g-tensor values of R2F-Mn^III₂-Tyr^•, estimated from X-band measurements, is poorly resolved in the HF-EPR spectrum. This is due to low radical concentration and the presence of manganese impurities overlapping on g₂ and g₃. HF-EPR is very sensitive to Mn(II) impurities and removal of most of the Mn(II) by gelfiltation was needed, which lower the tyrosyl-radical content in R2F-Mn^III₂-Tyr^• and at 5 K the two Mn(II) impurities (Figure S2) are partially saturated. At 10 K or 15 K the R2F-Mn^III₂-Tyr^• radical 285 GHz spectrum, is much more dominated by the Mn(II) impurities. Our findings indicate that presence of Mn(II) impurities can only be partly removed from the sample, but at the cost of a substantial loss in radical yield and resolution of the g-tensor.

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Figure 6. The X-band (9.66 GHz) EPR spectrum of R2F-Mn^III₂-Tyr^• B. cereus R2F (200 µM R2F) reconstituted in presence of 2X NrdI_hq.

Recorded at T = 20 K, 16 µW microwave power, 0.2 mT modulation amplitude, and 4 scans (Obs) and its spectrum simulation (Sim).

https://doi.org/10.1371/journal.pone.0033436.g006

In the Mn reconstitution process, the use of NrdI_ox was found to be ineffective, and only the use of NrdI_hq lead to successful loading of Mn into the protein active site competent for oxygen activation and formation of tyrosyl radical. The X-band double integrated EPR signal of R2F-Mn^III₂-Tyr^• followed the Curie-law and furthermore the spectra recorded at T = 50 K exhibited similar resonance envelope as those observed at lower temperatures without signal- broadening. The tyrosyl radical signal of R2F-Mn^III₂-Tyr^• shown in Figure 6 was obtained after EDTA treatment under anaerobic conditions of apo-R2F and followed by oxygen exposure before sample measurement. From a chemical perspective, the tyrosyl radical in R2F-Mn^III₂-Tyr^• appeared less stable than in R2F-Fe^III₂-Tyr^•, and the radical fingerprints (UV/Vis and EPR) degraded rapidly when the protein samples were not quickly frozen in liquid N₂. The overall radical X-band EPR resonance envelope has Curie-type temperature behavior and no modification of the EPR signal width from 4 K to 50 K in B. cereus R2F-Mn^III₂-Tyr^•, render the magnetic properties of the R2F-Mn^III₂-Tyr^• system in B. cereus different from those reported for other manganese forms, in E. coli and in C. ammoniagenes RNR R2F [20], [26]. In these later R2F proteins, the X-band EPR resonances changed significantly with the temperature, and contained a so-called “split-signal” arising from a strong magnetic interaction with the dinuclear Mn^III cluster that collapsed at T>30 K [20], [26]. Importantly, the total spectrum-width (X-band) in C. ammoniagenes RNR R2F was as large as 40 mT at T = 20 K, while in B. cereus the resonance envelope is less wide, at least by a four fold factor. Thus, the magnetic interaction between the tyrosyl radical and dimanganese catalytic core in B. cereus R2F must not be as strong as in E. coli and C. ammoniagenes R2Fs. The observed limited spectral broadening is consistent with the presence of a small, through-space, dipolar contribution. Even though the X-band EPR spectral simulation does not reproduce exactly the observed spectrum-width (Figure 6), the main resonance features are convincingly reproduced. The derived A_H terms for the tyrosine protons backbone and the overall EPR envelope suggest electronic and/or conformational changes in the tyrosyl radical in R2F-Mn^III₂-Tyr^• and slightly ∼5° θ (Table 1) larger than in R2F-Fe^III₂-Tyr^•. As described in the following section, such differences between the tyrosyl radical with nearby manganese or iron metal ions, appear also in the microwave power saturation behaviors of R2F-Mn^III₂-Tyr^• and R2F-Fe^III₂-Tyr^•, as observed by both the P_1/2 and b values.

Differences in X-band EPR microwave power saturation properties of tyrosyl radical in B. cereus R2F reconstituted with Fe or Mn

Analysis of progressive microwave power saturation of the EPR spectra at different temperatures, even without employment of relaxation enhancement in the saturation-recovery experiment, can provide insights into the relaxation properties of the radical system and its surrounding, as discussed in the seminal works of Portis [42], Castner [41] and Sahlin et al. [61], and well summarized recently by Hirsh and Brudvig [45]. The microwave power saturation results of R2F-Mn^III₂-Tyr^• and R2F-Fe^III₂-Tyr^• proteins were compared with those previously derived from different class Ia and Ib R2/R2F proteins. The microwave power saturation experiments are sensitive to interactions between a radical unit and a fast-relaxing metal site. The presence of effective magnetic interaction should change the radical relaxation properties, due to changes in T₁ (spin-lattice), T₂ (spin-spin) or both. P_1/2 and b become informative parameters in this case, b-values tend to converge to values smaller than 1 as a result of dipolar and exchange contributions (hence relaxation is neither purely homogeneous or purely inhomogeneous), while the half-saturation value (P_1/2), directly linked to 1/(T₁T₂), is expected to become larger than that observed in absence of magnetic interaction. The microwave power saturation trends observed at different temperatures for R2F-Fe^III₂-Tyr^• are shown in Figure 7A. The calculated b value is 1 (inhomogeneously broadened line) and the P_1/2 -value at T = 20 K (P_1/2 = 0.05 mW) was found lower than that obtained from B. anthracis R2F, M. tuberculosis R2F, and mouse R2, which all exhibit a P_1/2-value of ∼0.1 mW at T = 20 K [35], [56], [61]. However, the P_1/2- value of R2F-Fe^III₂-Tyr^• in B. cereus was slightly larger than in E. coli R2 (P_1/2 = 0.03 mW) at the same temperature [35], [61]. At higher temperatures (T = 50 K and T = 100 K), the P_1/2-values in B. cereus R2F becomes closer to those observed in the class Ib R2F proteins from M. tuberculosis [56] and B. anthracis [35]. In the higher temperature region, the class Ib R2F-FeIII₂-Tyr^• proteins all have lower P_1/2-values relative to class Ia R2's. This trend suggests a weak interaction between the tyrosyl radical and the diiron centre at high temperature. This weaker interaction can be rationalized by longer distance of the tyrosyl radical to the diiron-oxygen cluster in the class Ib proteins, which is observed in their crystal structures [37], [62], [63], [64], [65], [66]. The power saturation behavior of R2F-Mn^III₂-Tyr^• in presence of NrdI is shown in Figure 7B. The differences in the overall saturation properties are large compared to R2F-Fe^III₂-Tyr^•. The relaxation factor b was found to be <1 over the entire temperature range examined and best fit by b = 0.6. Attempts to fit the data by forcing constraint on b close to unity gave unreliable fits to the microwave power saturation data, thus the recorded trends with b = 0.6 indicate the presence of magnetic interaction between the radical and the dimanganese cluster, with a magnetic ground state. Because the overall radical line-width broadening of R2F-Mn^III₂-Tyr^• compared to R2F-Fe^III₂-Tyr^•, R2F-Mn^III₂-Tyr^• spectral shape did not change substantially at high temperatures and b appears constant and smaller than 1, further supporting a weak dipolar interaction of the radical unit with the ferromagnetic R2F-Mn^III₂ cluster. Our EPR signal broadening, as shown previously, was very small, and much smaller than that observed in the R2F-Mn^III₂ cluster of C. ammoniagenes. The different P_1/2 microwave power saturation between the iron and manganese reconstituted RNR R2 proteins are readily explained by weak ferromagnetic coupling of Mn^III₂ cluster as observed in C. ammoniagenes. In the R2F-Fe^III₂-Tyr^• system the effective anti-ferromagnetic coupling between the two ferric S = 5/2 centres [62] below 15 K render the magnetic properties of the tyrosyl radical well described as an isolated radical S = 1/2 spin system, since can not be perturbed by nearby effective (with S≠0) spin-systems [62]. On the contrary, the low b-factor determined for R2F-Mn^III₂-Tyr^• extrapolated in the entire temperature range (50–4 K) being accompanied by EPR signal broadening, implies that the tyrosyl radical interacts in B. cereus R2F with an additional spin-system (where S≠0) linked to the presence of the ferromagnetic di-manganese core. Consistent with our findings, the earlier work of Cox et al. and Cotruvo and Stubbe suggested the presence of a magnetic Mn^IIIMn^III cluster at low temperature for the R2F-Mn^III₂-Tyr^• [20], [26]. It is important to underline that the exact description of the spin multiplicity together with the size of the exchange-energy (J) terms among organic/metal spin centres are not yet clear in this system. Our P_1/2–value at 4 K (0.02 mW) clearly shows presence of interaction with a magnetic di-Mn cluster, while at this temperature with the di-Fe protein only show intrinsic tyrosyl-radical behavior without any magnetic interaction to an other center resulting in orders of magnitudes lower P_1/2–value. The P_1/2–values reported for the R2F-Mn^III₂-Tyr^• from E. coli Class Ib are quite different from those observed for B. cereus R2F, and have been found much larger in the former [20]. However, the large differences in the extrapolated b-factors do not allow direct comparison of these B. cereus R2F data with those obtained in R2F proteins from other sources. Our R2F-Mn^III₂-Tyr^• measurements, on one hand, were performed in presence of NrdI, which increases protein stability, and it is even possible to crystallize a R2F-NrdI complex [23]. On the other hand, the C. ammoniagenes and E. coli magnetic properties were studied as the R2F-Mn^III₂-Tyr^•without presence of NrdI. Therefore such differences in the samples chemical composition could explain the experimental variation of the R2F-Mn^III₂-Tyr^• magnetic properties among protein variants, as a result of protein-protein induced changes (e.g. in presence of NrdI) of the tyrosyl-radicals.

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Figure 7. The Microwave power saturation of the iron reconstituted tyrosyl radical in B. cereus R2F recorded at T = 20 K (blue circles), T = 50 K (violet circles) and at T = 100 K (orange circles) (A).

The factor ∫∫S represents the double integrated EPR signal intensity, P the applied microwave power and ∫∫S₀ the double integrated EPR signal intensity recorded at the lowest microwave power P₀. The correspondent simulations of the experimental data are shown by solid lines. The b-values were equal to 1 for T = 20 K and 50 K and b = 0.9 for T = 100 K. P_1/2 (20 K) = 0.05 mW, P_1/2 (50 K) = 0.3 mW, P_1/2 (100 K)≥2.6 mW. Panel (B) shows the microwave power saturation of the manganese reconstituted tyrosyl radical in B. cereus R2F recorded at T = 4 K (blue circles), T = 20 K (violet circles), T = 50 K (orange circles). The b-values were found equal to 0.6 for all the temperatures examined. P_1/2 (4 K) = 0.02 mW, P_1/2 (20 K) = 0.10 mW, P_1/2 (50 K) = 0.46 mW.

https://doi.org/10.1371/journal.pone.0033436.g007

Resonance Raman studies of B. cereus R2F reconstituted with Fe

Resonance Raman (rRaman) spectroscopy can be employed to unveil minor interactions of the tyrosyl radical with its molecular surrounding. Redox-linked structural changes associated with the electron transfer reaction can be elucidated using vibrational spectroscopy, EPR spectroscopy and by protein crystallography. For instance, tyrosyl radicals e.g., in E. coli RNR R2 (Y122^•) can show movements up to a 1 Å compared to their corresponding not oxidized tyrosine form (Y122) in E. coli RNR, S. typhimurium or mouse R2 and they have been proven to be coupled to a conformational change in the R2 subunit [65], [67], [68], [69]. The major vibrational fingerprint of the radical unit is the phenoxyl ν_7a band (Wilson notation) [70], which contains a major contribution from a C-O^• stretching vibration and is a sensitive marker for hydrogen bonding, see our recent study of R2 tyrosyl radical from Epstein-Barr virus [71]. This Raman mode is strongly resonance enhanced by excitation frequencies (λ_ex) around 405–415 nm, close to the Tyr^• absorption maximum (∼410 nm) as shown in Figure 2. The ν_7a mode is observed at 1497–1501 cm⁻¹ in absence of a hydrogen bond interaction, e.g. E. coli R2 [72], while in mouse R2, this value shifts at 1515 cm⁻¹ due to hydrogen bonding [40] (Table 2). In mouse R2 a rather close (-O^…H = 1.89 Å) proton (exchangeable with D₂O) is present near the tyrosyl oxygen, which was observed with Davies ENDOR experiments [40], [55]. The tyrosyl radical excitation rRaman spectrum of B. cereus R2F-Fe^III₂-Tyr^• (Figure 8, black spectrum) has a peak at 1500 cm⁻¹ (λ_exc = 407 nm).This Raman mode remains unchanged upon deuterium-oxide exchange (blue spectrum) supporting that no hydrogen bond to the phenoxyl group of the tyrosyl radical is present. Furthermore, theoretical calculation (UB3LYP/6-311++G(d,p)) carried out on the tyrosyl radical under constrained dihedral angle (θ = 60°) well reproduces the experimentally determined energy position of the C-O^• vibration (ν_7a calc = 1495 cm⁻¹) (Calculation S1).

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Figure 8. Resonance Raman spectra of the active form of Fe-reconstituted B. cereus R2F form.

The resonance signature of the Fe-O-Fe vibration region is shown in the left side and the tyrosyl radical C-O vibration is shown in the right side of the panel. Protein recorded in H₂O (black line) and after isotopic substitutions with D₂O (blue line) or H₂¹⁸O (red line). Methods for isotope substitution are described in the Materials Section. The left side of the panel shows with A) the lower energy region recorded with λ_exc = 379.5 nm, 5 mW at the sample and T = 77 K and B) λ_exc = 410 nm, 5 mW at the sample and T = 77 K. The right side of the panel shows the higher energy region recorded with λ_exc = 406.7 nm, 10 mW at the sample and T = 77 K.

https://doi.org/10.1371/journal.pone.0033436.g008

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Table 2. Resonance Raman parameters for the Fe(III)-O-Fe(III) protein sites and phenoxyl ν_7a bands of different RNR proteins.

https://doi.org/10.1371/journal.pone.0033436.t002

The low frequency region of the rRaman spectrum can provide valuable information about the ferric Fe-O-Fe cluster. Proteins with a bent μ-oxo-bridged diferric center have characteristic UV/Vis absorption bands in the 300–450 nm region that are not observed in corresponding μ-hydroxo-bridged proteins or model compounds [7]. The symmetric Fe-O-Fe stretching mode observed around 483 cm⁻¹ (Figure 8, black line) is coupled to the oxygen to iron charge transfer transition [7]. This mode displays an ¹⁸O- induced shift to lower energy in the rRaman spectrum. In the low frequency region (below 1000 cm⁻¹) of the active B. cereus R2F-Fe^III₂ rRaman spectrum, with λ_exc at 364 nm or 407 nm, a symmetric vibration at 483 cm⁻¹ is observed. This vibrational band is lacking in the rRaman spectra of the apo protein under similar conditions. When the protein was exchanged into H₂¹⁸O-water and reconstituted with ¹⁸O (g), a −14 cm⁻¹ shift emerged (Figure 8, red line). The 483 cm⁻¹ band could therefore be assigned to the symmetric Fe^III-O-Fe^III stretching mode. Table 2 illustrates a comparison of the Fe-O-Fe vibrations and the phenoxyl ν_7a modes for different RNR proteins. The Fe^III-O-Fe^III stretching mode occurs 12 cm⁻¹ lower than in E. coli R2 and 3 cm⁻¹ lower than in mouse R2 suggesting weaker mechanical coupling between the two ferric irons. No change in the Fe-O-Fe vibration was observed in the low energy region of the rRaman spectrum when B. cereus R2F was exchanged into D₂O-containing buffer (Figure 8, blue line). Deuterium oxide incubated mouse R2 has a small 5 cm⁻¹ downshift in the frequency associated with the Fe-O-Fe vibration that indicates the presence of a hydrogen bond to the μ-oxo bridge [40], thus we have no indication of similar hydrogen bond in B. cereus R2F. It has been suggested that the hydrogen bond to the μ-oxo bridge could have an influence on the formation of a stable mixed-valence (Fe^IIIFe^II) cluster. The mixed valence form has been observed by EPR for mouse and herpes simplex virus R2 but not in E. coli R2 or B. cereus R2F [40], [73], [74]. Presently, we were unable to obtain resolved rRaman spectra of the R2F-Mn^III₂-Tyr^• form, due to the low radical concentration, stability that the reconstituted protein sample exhibits and presence of the flavin in NrdI with very strong background.

Conclusion

We have used UV/Vis, X-band, HF-EPR, and rRaman to investigate iron and manganese reconstituted tyrosyl radicals from B. cereus R2F. The X-band EPR signal of the tyrosyl radical reconstituted with iron or manganese exhibits some features that are notably different. In R2F-Fe^III₂-Tyr^•, the properties of the tyrosyl radical can be described in terms of a tyrosyl radical not broadened by the iron-cluster below 50 K, only changing the relaxation properties. However, in R2F-Mn^III₂-Tyr^•, the dipolar magnetic interactions between the Tyr^• centre and the ferromagnetic manganese- cluster are effective over the whole temperature range examined (from 4 K to 50 K), broadening the tyrosyl radical spectrum and inducing a low b-value and high P_1/2. The EPR resonance fingerprint and temperature behavior of R2F-Mn^III₂-Tyr^• are different from those observed in the Mn forms of E. coli, C. ammoniagenes and the recently discovered B. subtilis [22].This argues for a weaker magnetic interaction between radical and metal sites in B. cereus, relative to those in E. coli and C. ammoniagenes RNR R2 proteins. Differences between the two metal reconstituted forms in B. cereus are also reflected in the spread of the g-tensor values as well as in the hfc with the tyrosyl-radical protons. In addition, we suggest the torsional angle θ slightly increases to 65° in R2F-Mn^III₂-Tyr^• as compared to R2F-Fe^III₂-Tyr^• (estimated as 60°). The two forms of reconstituted RNR R2 proteins show differences in chemical stability, with R2F-Fe^III₂-Tyr^• being most stable. Furthermore, the use of NrdI_ox in the Mn reconstitution process was ineffective; only when NrdI_hq was present in the sample, the RNR R2 protein could be successfully reconstituted with Mn. HF-EPR and rRaman studies of the R2F-Fe^III₂-Tyr^• form suggest that the radical is not involved in hydrogen bonding interactions with nearby groups (high g₁ value and ν_7a = 1500 cm⁻¹). The specific activities of the Mn-O-Mn core in R2F proteins have been tested recently in both B. cereus and B. anthracis. The studies evidenced that these systems behaved similarly, showing a raise in protein activity (about eight times) when manganese ions occupy the di-nuclear binding site instead of iron and in presence with the full natural disulfide reductant system [38], [75], [76]. The B. cereus R2F-Mn^III₂-Tyr^• form shows high dependence for the natural R1E reductant NrdH (BC3987 a thioredoxin protein) during activity measurement [38], [75], [76]. These findings reinforce the hypothesis that R2F-Mn^III₂-Tyr^• form is biologically functional and under some conditions the other Fe-O-Fe form may become active within cell metabolism as well. Our study provides a further example of the spread of finely tuned electronic/magnetic properties associated with both the tyrosyl radical and the catalytic/metal-oxygen binding site present in RNR R2 class Ib proteins.