The proton dependence of nicotinate binding to soybean, kidney bean and lupin ferric leghemoglobins

Abstract

Abstract The binding affinities of nicotinate, nicotinamide and pyridine for soybean ferric leghemoglobin a, kidney bean ferric leghemoglobin a and lupin ferric leghemoglobin I were measured over the pH range of 4.0–10.5. For all proteins the nicotinate affinity increased with lowering of pH, but the nicotinamide and pyridine affinities were pH-independent. For lupin leghemoglobin I, the nicotinate-binding affinity curve revealed a probable single ionization of pKa of 6.4, tentatively ascribed to the distal histidine residue E7. For soybean and kidney bean leghemoglobins a, the nicotinate binding curves can be separated into two ionization steps with pKa,1 of 4.9 and pKa,2 of 7.7 (soybean) and pKa,1 of 4.7 and pKa,2 of 7.5 (kidney bean). The alkaline ionizations aae tentatively ascribed to distal histidine and the acid ionizations to glutamate E9, the latter supposedly acting as an electrostatic gate against anion binding. Only steric factors appear to influence binding of the uncharged molecules nicotinamide and pyridine.