Figure 1.
The Mn-substituted R2F-protein from C. ammoniagenes (PDB code 3MJO) [26] (A). and sketch of themolecular structure of the tyrosyl radical (B).
In panel (A) the numbers in bold represent atomic distances (Å). The structure in (B) describes the dihedral angle θ (C6-C1-Cβ-Cα) used to illustrate the orientation of the α-carbon: θ = 0° corresponds to the α-carbon in the plane of the phenoxyl ring, while θ = 90° is perpendicular to the plane.
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 YD radical in photosystem II, *Inequivalent C3 and C5 protons, **Using XSophe (v. 1.1.4) (matrix diagonalization, g-strain model).
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 MnII2-R2F reconstituted with 25 µM of NrdIhq and O2 (g) (blue line), 50 µM MnII2-R2F with 25 µM of NrdIox (dark-yellow line), and manganese reconstituted R2F treated with 4 mM HU for 5 min at room temperature (red line).
Figure 3.
X-band (9.66 GHz) EPR spectrum of the R2F-FeIII2-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).
Figure 4.
HF-EPR (285 GHz) spectrum of R2F-FeIII2-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).
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.
Figure 6.
The X-band (9.66 GHz) EPR spectrum of R2F-MnIII2-Tyr• B. cereus R2F (200 µM R2F) reconstituted in presence of 2X NrdIhq.
Recorded at T = 20 K, 16 µW microwave power, 0.2 mT modulation amplitude, and 4 scans (Obs) and its spectrum simulation (Sim).
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. P1/2 (20 K) = 0.05 mW, P1/2 (50 K) = 0.3 mW, P1/2 (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. P1/2 (4 K) = 0.02 mW, P1/2 (20 K) = 0.10 mW, P1/2 (50 K) = 0.46 mW.
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 λexc = 379.5 nm, 5 mW at the sample and T = 77 K and B) λexc = 410 nm, 5 mW at the sample and T = 77 K. The right side of the panel shows the higher energy region recorded with λexc = 406.7 nm, 10 mW at the sample and T = 77 K.
Table 2.
Resonance Raman parameters for the Fe(III)-O-Fe(III) protein sites and phenoxyl ν7a bands of different RNR proteins.