Nitric oxide synthase (NOS) catalyzes the formation of NO and citrulline from L-arginine and oxygen. However, the NO so formed has been found to auto-inhibit the enzymatic activity significantly. We hypothesized that the NO reactivity is, in part, controlled by hydrogen bonding between the conserved tryptophan residue (position 409 in nNOS) and the cysteine residue that forms the proximal bond to the heme. By using resonance Raman spectroscopy and NO as a probe of the heme environment, we show that in the W409F and W409Y mutants of the oxygenase domain of the neuronal enzyme (nNOSox), the Fe-NO bond in the Fe³⁺NO complex is weaker than in the wild type enzyme, consistent with the loss of a hydrogen bond on the sulfur atom of the proximal cysteine residue. The weaker Fe-NO bond in the W409F and W409Y mutants might result in a faster rate of NO dissociation from the ferric heme in the W409 mutants, as compared to the wild type enzyme, which could contribute to the lower accumulation of the inhibitory NO-bound complexes observed during catalysis with the W409 mutants (Adak et al. 1999. J. Biol. Chem. 274, 26907-26911). The optical and resonance Raman spectra of the Fe²⁺NO complexes of the W409 mutants differ from those of the wild type enzyme and indicate that a significant population of a five-coordinate Fe²⁺NO-complex is present. These data show that the hydrogen bond provided by the W409 residue is necessary to maintain the thiolate coordination when NO binds to the ferrous heme. Taken together our results indicate that the heme environment on the proximal side of nNOS is critical for the formation of a stable Fe-cysteine bond and for the control of the electronic properties of heme-NO complexes.