Computational studies on aspartic proteases. I. Active-site protonation and hydration in the substrate-free crystalline state

We performed semiempirical molecular orbital calculations on the free and hydrated states of the HCOO^?…HCOOH couple in crystals of an aspartic protease, rhizopuspepsin, in order to gain information not available by conventional X-ray diffraction studies. The reliability of the MNDO /PM 3 method was proven for the model system HCOO^?…HOH, for which we could reproduce geometry and energetics of the complex, obtained by sophisticated ab initio calculations, with astonishing accuracy. Comparing the geometries of the active-site models to experiment and their relative energies, we suggest that in the substrate-free crystalline state the carboxyl–carboxylate–hydroxonium triad exists most probably in the neutral form. The carboxyl–carboxylate dyad attracts a hydroxonium ion better than does ammonium, indicating that the nonhydrogen atom, located by X-ray crystallography near the active site, is oxygen and not nitrogen. We located the most probable hydrogen positions by geometry optimization.