Nitric oxide synthases (NOS) are a family of cysteine thiolate-ligated heme-containing monooxygenases that catalyze the NADPH-dependent two-step conversion of L-arginine to NO and L-citrulline. During the catalysis, a portion of the NOS heme forms an inhibitory complex with self-generated NO that is subsequently reverted back to NO-free active enzyme under aerobic conditions, suggesting a downstream regulator role of NO. Recent studies revealed that mutation of a conserved proximal tryptophan-409, which forms one of three hydrogen bonds to the heme-coordinated cysteine thiolate, to tyrosine or phenylalanine considerably increases the turnover number of neuronal NOS (nNOS). To further understand these properties of nNOS on its active site structural level, we have examined the oxygenase (heme-containing) domain of the two mutants in close comparison with that of wild-type nNOS with UV - visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy. Among several oxidation and ligation states examined, only the ferrous - NO adducts of the two mutants exhibit spectra that are markedly distinct from those of parallel derivatives of the wild-type protein. The spectra of the ferrous - NO mutants are broadly similar to those of known five-coordinate ferrous - NO heme complexes, suggesting that these mutants are predominantly five coordinate in their ferrous - NO states. The present results are indicative of cleavage of the Fe - S bond in the nNOS mutants in their ferrous - NO state and imply a significant role of the conserved tryptophan in stabilization of the Fe - S bond.
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