Nitric oxide synthases (NOSs) are heme proteins that catalyze the formation of nitric oxide from L-Arg in the presence of oxygen. Of the two electrons required for the first step of the reaction, the second is primarily donated by the tetrahydrobiopterin (H4B) cofactor bound adjacent to the heme, which is eventually reduced back to resting state by the ultimate electron source of the reaction, the flavins of the NOS reductase domain. Density functional theory calculations were carried out to identify those protonation states of different cofactor molecules that best support radicalization of the cofactor and the coupled increase in the electron density of the herne-bound oxygen molecule. Three cofactor molecules were studied, native H4B, an active analogue, 5-methyl-H4B, and the inactive 4-amno-H4B. Findings support the emerging model where H4B and 5-methyl-H4B are coupled proton/electron sources of NOS catalysis, while 4-amino-H4B is an inhibitor due to its inability to donate the catalytically required proton.