Abstract
Nitric oxide (NO) produced by mammalian nitric oxide synthases (mNOSs) is an important mediator in a variety of physiological functions. Crystal structures of mNOSs have shown strong conservation of the active‐site residue Val567 (numbering for rat neuronal NOS, nNOS). NOS‐like proteins have been identified in several bacterial pathogens, and these display striking sequence identity to the oxygenase domain of mNOS (NOSoxy), with the exception of a Val to Ile mutation at the active site. Preliminary studies have highlighted the importance of this Val residue in NO‐binding, substrate recognition, and oxidation in mNOSs. To further elucidate the role of this valine in substrate and substrate analogue recognition, we generated five Val567 mutants of the oxygenase domain of the neuronal NOS (nNOSoxy) and used UV‐visible and EPR spectroscopy to investigate the effects of these mutations on the heme distal environment, the stability of the heme‐FeII‐CO complexes, and the binding of a series of substrate analogues. Our results are consistent with Val567 playing an important role in preserving the integrity of the active site for substrate binding, stability of heme‐bound gaseous ligands, and potential NO production.
Highlights
Nitric oxide (NO) produced by mammalian nitric oxide synthases is an important mediator in a variety of physiological functions
High levels of NO are involved in several pathologies associated with oxidative stress phenomena such as atherosclerosis, Abbreviations BH4, (6R)-5,6,7,8 tetrahydro-L-biopterin; DTT, dithiothreitol; ImH, imidazole; L-Arg, L-arginine; mNOS, mammalian NOS; nNOS, neuronal NOS; NOHA, Nx-hydroxy-L-arginine; NOS, nitric oxide synthase; NOSoxy, oxygenase domain of NOS; PMSF, phenylmethylsulfonyl fluoride; WT, wild-type
WT nNOSoxy, and its Val567Ser, Val567Tyr, and Val567Thr mutants were purified in almost identical yield (3–5 mgÁLÀ1 of culture), and Val567Phe was obtained in a slightly higher yield (6–8 mgÁLÀ1 of culture), whereas Val567Arg was obtained in a lower yield (1–2 mgÁLÀ1 of culture)
Summary
Nitric oxide (NO) produced by mammalian nitric oxide synthases (mNOSs) is an important mediator in a variety of physiological functions. The structure of the three mNOS isoforms can be divided into two main parts: a N-terminal oxygenase domain (NOSoxy) containing the heme prosthetic group, with binding sites for substrate L-arginine (L-Arg), and cofactor (6R)-5,6,7,8-tetrahydro-L-. Biopterin (BH4), and a C-terminal reductase domain containing binding sites for cofactors, FMN, FAD, and NADPH. High levels of NO are involved in several pathologies associated with oxidative stress phenomena such as atherosclerosis, Abbreviations BH4, (6R)-5,6,7,8 tetrahydro-L-biopterin; DTT, dithiothreitol; ImH, imidazole; L-Arg, L-arginine; mNOS, mammalian NOS; nNOS, neuronal NOS; NOHA, Nx-hydroxy-L-arginine; NOS, nitric oxide synthase; NOSoxy, oxygenase domain of NOS; PMSF, phenylmethylsulfonyl fluoride; WT, wild-type.
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