Nitric oxide is synthesized from L-Arg by nitric oxide synthases (NOSs). DFT calculations carried out in the present study demonstrate that there is direct coupling between the heme bound oxygen and the tetrahydrobiopterin (H4B) cofactor in the activated state of NOS. Results indicate that radicalization of H4B causes the coupled reduction of heme bound oxygen. In our model system H3B. radical formation is prompted by proton dissociation from the N5 site of the cofactor; spin density is transferred to the heme bound oxygen, which we found in an orientation preconditioned for H abstraction from the substrate. (C) 2004 Elsevier B.V. All rights reserved.