The catalytic center of nitric oxide synthase (NOS) consists of a thiolate coordinated heme macrocycle, a tetrahydrobiopterin (H4B) cofactor and an L-arginine (L-Arg) / N-hydroxy-L-arginine (NOHA) substrate binding site. To determine how the interplay between the cofactor, the substrates, and the protein matrix housing the heme regulates the enzymatic activity of NOS, the CO, NO, and CN-bound adducts of the oxygenase domain of the inducible isoform of NOS (iNOSoxy) were examined with resonance Raman spectroscopy. The Raman data of the CO-bound ferrous protein demonstrated that the presence of L-Arg causes the Fe-C-O moiety to adopt a bent structure due to an H-bonding interaction whereas H4B binding exerts no effect. Similar behavior was found in the CN-bound ferric protein and in the NO-bound ferrous protein. In contrast, in the NO-bound ferric complexes, the addition of L-Arg alone does not affect the structural properties of the Fe-N-O moiety, but H4B binding forces it to adopt a bent structure, which is further enhanced by the subsequent addition of L-Arg. The differential interactions between the various heme ligands and the protein matrix in response to L-Arg and/or H4B binding is coupled to heme distortions, as reflected by the development of a variety of out-of-plane heme modes in the low frequency Raman spectra. The extent and symmetry of heme deformation, modulated by ligand, substrate and cofactor binding, may provide important control over the catalytic and auto-inhibitory properties of the enzyme.