Demonstration of a conserved histidine and two water ligands at the Mn2+ site in diocleinae lectins by pulsed EPR spectroscopy

Lectins from the Diocleinae subtribe, including Canavalia brasiliensis, Can avalia bonariensis, Canavalia grandiflora, Cratylia floribunda, Dioclea gra ndiflora, Dioclea guianensis, Dioclea rostrata, Dioclea violacea, and Diocl ea virgata, have been recently isolated and characterized in terms of their carbohydrate binding specificities. Although all of the lectins are Man/Gl c specific, they possess different biological activities. In the present st udy, electron paramagnetic resonance (EPR) spectroscopy demonstrates that a ll nine Diocleinae lectins contain Mn2+. The spectra of C. floribunda and D . rostrata suggest Mn2+ site symmetry different from that of the other seve n lectins. However, electron spin-echo envelope modulation (ESEEM) spectros copy indicates that all nine lectins are coordinated to a histidyl imidazol e, with similar electron-nuclear coupling to the Mn2+-bound imidazole nitro gen. ESEEM also demonstrates ligation of two water molecules to Mn2+ in all nine Diocleinae lectins. Thus, the EPR and ESEEM data indicate the presenc e of a Mn2+ binding site in the above Diocleinae lectins with a conserved h istidine residue and two water ligands.