A charge polarization model for the metal-specific activity of superoxide dismutases

Abstract

The pathogenicity of Staphylococcus aureus is enhanced by having two superoxide dismutases (SODs): a Mn-specific SOD and another that can use either Mn or Fe. Using 94 GHz electron-nuclear double resonance (ENDOR) and electron double resonance detected (ELDOR)-NMR we show that, despite their different metal-specificities, their structural and electronic similarities extend down to their active-site ¹H- and ¹⁴N-Mn(ii) hyperfine interactions. However these interactions, and hence the positions of these nuclei, are different in the inactive Mn-reconstituted Escherichia coli Fe-specific SOD. Density functional theory modelling attributes this to a different angular position of the E. coli H171 ligand. This likely disrupts the Mn-H171-E170' triad causing a shift in charge and in metal redox potential, leading to the loss of activity. This is supported by the correlated differences in the Mn(ii) zero-field interactions of the three SOD types and suggests that the triad is important for determining metal specific activity.

Fig. 1. The structure of the interconnected metal sites of the two subunits of Mn/FeSODs, with carbon atoms in green, oxygen atoms in red, nitrogen atoms in blue, and the metal ion in purple. The residue numbering here, and in the text, is based on the familiar E. coli MnSOD (see the ESI†).

Fig. 2. The energy level diagram (top) and geometric arrangement (bottom) of a Mn(ii, S = 5/2) coupled to protons (I = 1/2) and nitrogens (I = 1). On the top, blue arrows correspond to electron spin transitions; the cyan lines, the NMR transitions measured by ENDOR; and magenta and purple lines are those measured by single- and double-quantum ELDOR-NMR. On the bottom, in red, the geometry of the hyperfine interaction between the Mn(ii) and a water ligand proton with the magnetic-field (B₀) applied along the Mn–N_ε,His26 bond (green) and, in purple, the orientation of the zero-field interaction axes (D_xx,yy,zz) with respect to the Mn(ii) ligands. H81 has been omitted for clarity.

Fig. 3. Graphic representation of Mn coordination in the active sites of the homodimers of S. aureus (A) MnSOD (ribbon in blue) and (B) camSOD (ribbon in teal). Mn is represented as purple spheres. The orange mesh represents the anomalous difference map rendered at 6.0σ and 15.0σ on the Mn ions at the active site of MnSOD and camSOD, respectively. Metal-coordinating ligand residues are represented as sticks with carbon atoms colored in grey, oxygens in red, nitrogens in blue and a water molecule represented as a red sphere (PDB accession codes: ; 5N56 for MnSOD and ; 5N57 for camSOD).

Fig. 4. 94 GHz 6 K Mn(ii) field-swept echo EPR spectra of: Mn(Mn)SOD (black); Mn(cam)SOD (green); and Mn(Fe)SOD (red). The indicated zero-field field positions are relative to ν_obs/gβ. Arrows indicate the magnetic-field positions where the ENDOR and ELDOR-NMR spectra were taken (their colors corresponding to the proteins).

Fig. 5. The 94 GHz 5 K SQ (bottom) and DQ (top) ¹H ELDOR-NMR spectra of Mn(Mn)SOD (black), Mn(cam)SOD (green) and Mn(Fe)SOD (red) taken at the D_zz,–5/2 field positions. The SQ spectra taken at the D_xx,–5/2 field position are shown in the inset. The exact field positions are indicated by the arrows in Fig. 2. The Gaussian line shape simulations of the SQ spectra are also shown, with their colors corresponding to the measured spectra and their sums as dashed lines. The DFT hyperfine histograms of the GO (black) and CD(NH) (red) models are superimposed and their correspondence to the measured spectra is indicated by the dotted-lines. For the D_zz,–5/2 spectra, ν_¹H,NMR was 79 MHz and for the D_xx,–5/2, it was 185 MHz. See text and the ESI for details.

Fig. 6. The 94 GHz 5 K Davies ¹H ENDOR spectra of Mn(Mn)SOD, Mn(Fe)SOD and Mn(cam)SOD taken at the D_yy,–3/2 magnet field positions indicated by the arrows in Fig. 2. The blue trace was obtained under the same conditions but at the D_–xx,–5/2 magnetic-field position indicated by the black arrow in Fig. 2. The initial electron-spin inversion pulse was 200 ns, followed by a 16 μs radio-frequency pulse and standard spin-echo detection (12 and 24 ns pulses). The lower panel shows the calculated ENDOR spectra based on DFT hyperfine tensors obtained for the GO (red) and CD(NH) (black) models. The cyan traces show the calculated spectra obtained by symmetrizing the DFT hyperfine tensors and manually adjusting the A_iso values. The calculated spectra have been convolved using 150 kHz Gaussian and each arbitrarily scaled (see text and the ESI for details).

Fig. 7. The 94 GHz 5 K SQ (bottom) and DQ (top) ¹⁴N ELDOR-NMR spectra of Mn(Mn)SOD (black), Mn(cam)SOD (green) and Mn(Fe)SOD (red) taken at the D_zz,–5/2 field positions indicated by the arrows in Fig. 2. The DFT hyperfine histograms of the GO (black) and CD(NH) (red) models (ν_¹⁴N,NMR = 5.7 MHz).

Fig. 8. The DFT GO, CD(NH) and CD(OH) model structures. The numbers indicate the CM5 charges of each atom of the His171 ring with their hydrogen charges, if any, summed into them. The orientation of the ligands in Mn(Mn)SOD and Mn(cam)SOD is shown in Fig. 3 and 6.