What form of NG-hydroxy-L-arginine is the intermediate in the mechanism of NO synthase? QM and QM/MM calculations of substrate-active site interactions

Different isomeric species of the intermediate N G-hydroxy-L-arginine (NHA) in the reaction of nitric oxide synthase (NOS) have been theoretically investigated in terms of their geometries and energetics, taking into account at a quantum mechanical level the hydrogen bonding network involved in the substrate site. A variety of models were used, including a hybrid QM/MM treatment of the complete enzyme. It was found that inclusion of the residues, local to the substrate, was crucial, particularly in predicting the energetic features of the species studied. Taking the enzyme into account we conclude that the zwitterionic form of NHA should not be ruled out as a possible intermediate together with neutral and protonated forms and that whilst O- and N-radicals are close in energy in the free form the inclusion of the enzyme strongly favours O-radicals. © 2003 Elsevier B.V. All rights reserved.