W-band ENDOR investigation of the manganese-binding site of concanavalin A: Determination of proton hyperfine couplings and their signs

W-band (95 GHz) pulsed EPR acid electron-nuclear double resonance spectroscopic: techniques were used to investigate the manganese S1 binding site of the protein concanavalin A in a frown solution and in a single crystal. H-1 ENDOR spectra were collected by using the Davies ENDOR sequence, whereas Mims ENDOR was applied to record H-2 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 similar to 3.4 T at low temperatures, allowed the assignment of ENDOR signals to their respective M-S manifolds, thus providing the sign of the hyperfine couplings. The exchangeable and nonexchangeable protons were differentiated by comparing H-1 and H-2 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 Angstrom 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(parallel to) and A(perpendicular to) were determined. All possible combinations of these values yield distances in the range of 2.67 to 3.24 Angstrom and a(iso) between -1.13 and 1.37 MHz. By limiting a(iso) to positive values, only four distances remain. 2.67, 2.76, 2.99, and 3.24 Angstrom, corresponding to the four Mn-H-water distances.