In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric oxide synthases (NOSs), we have cloned, expressed and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from L-arginine via the stable intermediate N-hydroxy L-arginine (NOHA). Addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates L-arginine and NOHA. The substrate induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO forms only in the presence of H4B. The crystal structure of gsNOS at 3.2 Å resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys356 (B. subtilis NOS) to Arg365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue closer to the heme. Thus, a more constrained heme-pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 °C. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation.