Nitrile reductases catalyse a two-step reduction of nitriles to amines. This requires the binding of two NADPH molecules during one catalytic cycle. For the nitrile reductase from E. coli (EcoNR) mass spectrometry studies of the catalytic mechanism were performed. EcoNR is dimeric and has no Rossman fold. It was demonstrated that during catalysis each active site binds one substrate molecule. NADPH binds independent of the substrate. The PreQ0 binding pocket of the active site is not involved in the binding of NADPH; this is in conflict with an earlier hypothesis.