W-Band ENDOR Investigation of the Manganese-Binding Site of Concanavalin A: Determination of Proton Hyperfine Couplings and Their Signs

Abstract

W-band (95 GHz) pulsed EPR and electron−nuclear double resonance spectroscopic techniques were used to investigate the manganese S1 binding site of the protein concanavalin A in a frozen solution and in a single crystal. 1H ENDOR spectra were collected by using the Davies ENDOR sequence, whereas Mims ENDOR was applied to record 2H ENDOR spectra. Different EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at magnetic fields of ∼3.4 T at low temperatures, allowed the assignment of ENDOR signals to their respective MS manifolds, thus providing the sign of the hyperfine couplings. The exchangeable and nonexchangeable protons were differentiated by comparing 1H and 2H ENDOR spectra of a solution of the protein prepared in D2O and of crystals soaked in D2O. The two imidazole protons, located on the carbons flanking the Mn-bound nitrogen, are magnetically equivalent, situated 3.56 Å from the Mn2+ and their hyperfine coupling is purely dipolar. The four protons of the two water ligands are all inequivalent, and four values of A∥ and A⊥ were determined. All possible combinations of these values yield distances in the range of 2.67 to 3.24 Å and aiso between −1.13 and 1.37 MHz. By limiting aiso to positive values, only four distances remain, 2.67, 2.76, 2.99, and 3.24 Å, corresponding to the four Mn−Hwater distances.