ENDOR and ESEEM investigation of the Ni-containing superoxide dismutase

Superoxide dismutases (SODs) protect cells against oxidative stress by disproportionating O₂ ⁻ to H₂O₂ and O₂. The recent finding of a nickel-containing SOD (Ni-SOD) has widened the diversity of SODs in terms of metal contents and SOD catalytic mechanisms. The coordination and geometrical structure of the metal site and the related electronic structure are the keys to understanding the dismutase mechanism of the enzyme. We performed Q-band ¹⁴N,^1/2H continuous wave (CW) and pulsed electron–nuclear double resonance (ENDOR) and X-band ¹⁴N electron spin echo envelope modulation (ESEEM) on the resting-state Ni-SOD extracted from Streptomyces seoulensis. In-depth analysis of the data obtained from the multifrequency advanced electron paramagnetic resonance techniques detailed the electronic structure of the active site of Ni-SOD. The analysis of the field-dependent Q-band ¹⁴N CW ENDOR yielded the nuclear hyperfine and quadrupole coupling tensors of the axial N_δ of the His-1 imidazole ligand. The tensors are coaxial with the g-tensor frame, implying the g-tensor direction is modulated by the imidazole plane. X-band ¹⁴N ESEEM characterized the hyperfine coupling of N_ε of His-1 imidazole. The nuclear quadrupole coupling constant of the nitrogen suggests that the hydrogen-bonding between N_ε–H and O_Glu-17 present for the reduced-state Ni-SOD is weakened or broken upon oxidizing the enzyme. Q-band ¹H CW ENDOR and pulsed ²H Mims ENDOR showed a strong hyperfine coupling to the protons(s) of the equatorially coordinated His-1 amine and a weak hyperfine coupling to either the proton(s) of a water in the pocket at the side opposite the axial N_δ or the proton of a water hydrogen-bonded to the equatorial thiolate ligand.