The high-potential flavin and heme of nitric oxide synthase are not magnetically linked: implications for electron transfer

Abstract Background: The homodimeric nitric oxide synthase (NOS) catalyzes conversion of l-arginine to l-citrulline and nitric oxide. Each subunit contains two flavins and one protoporphyrin IX heme. A key component of the reaction is the transfer of electrons from the flavins to the heme. The NOS gene encodes two domains linked by a short helix containing a calmodulin-recognition sequence. The reductase domain binds the flavin cofactors, while the oxygenase domain binds heme and l-arginine and additionally mediates the dimerization of the NOS subunits. We investigated the origin of the unusual magnetic properties (rapid-spin relaxation) of an air-stable free radical localized to a reductase domain flavin cofactor. Results: We characterized the air-stable flavin in wild-type NOS, both in the presence and absence of calcium and calmodulin, the imidazole-bound heme complex of wild-type NOS, the NOS Cys415→Ala mutant, and the isolated reductase domain. All preparations of NOS had the same flavin electron-spin relaxation behavior. No half-field transitions or temperature-dependent changes in the linewidth of the radical spin signal were detected. Conclusions: These data suggest that the observed relaxation enhancement of the NOS flavin radical is caused by the environment provided by the reductase domain. No magnetic interaction between the heme and flavin cofactors was detected, suggesting that the flavin and heme centers are probably separated by more than 15 A.