Dynamics in the Mn2+ binding site in single crystals of concanavalin a revealed by high-field EPR spectroscopy

EPR spectroscopy at 95 GHz was used to characterize the dynamics at the Mn2+ binding site in single crystals of the saccharide-binding protein concanavalin A. The zero-field splitting (ZFS) tensor of the Mn2+ was determined from rotation patterns in the a-c and a-b crystallographic planes, acquired at room temperature and 4.5 K. The analysis of the rotation patterns showed that while at room temperature there is only one type of Mn2+ site, at low temperatures two types of Mn2+ sites, not related by any symmetry, are distinguished. The sites differ in the ZFS parameters D and E and in the orientation of the ZFS tensor with respect to the crystallographic axes. Temperature-dependent EPR measurements on a crystal oriented with its crystallographic a axis parallel to the magnetic field showed that as the temperature increases, the two well-resolved Mn2+ sextets gradually coalesce into a single sextet at room temperature. The line shape changes are characteristic of a two-site exchange. This was confirmed by simulations which gave rates in the range of 10(7)-10(8) s(-1) for the temperature range of 200-266 K and an activation energy of 23.8 kJ/mol. This dynamic process was attributed to a conformational equilibrium within the Mn2+ binding site which freezes into two conformations at low temperatures.