Four possible reaction paths of an Fe-containing nitrile hydratase (NHase) are examined using the method of hybrid quantum mechanics/molecular mechanics (QM/MM) to reveal the initial steps of the catalytic mechanism of NHases. The reaction barrier for a nucleophilic attack of the sulfenic oxygen of alpha Cys114-SO- to the substrate is the lowest among the four paths, though another path, in which a water molecule activated by alpha Cys114-SO- attacks the substrate, also has a low enough barrier. Under the protein environment, beta Arg56 has weaker proton donor ability and shows no proton transfer to alpha Cys114-SO-, which was reported in model clusters. (C) 2015 Elsevier B.V. All rights reserved.