Characterization by electron paramagnetic resonance of the interactions of L-arginine and L-thiocitrulline with the heme cofactor region of nitric oxide synthase.
Nitric oxide synthase (NOS) catalyzes sequential NADPH- and O2-dependent mono-oxygenase reactions converting L-arginine to N omega-hydroxy-L-arginine and N omega-hydroxy-L-arginine to citrulline and nitric oxide. The homodimeric enzyme contains one heme/monomer, and that cofactor is thought to mediate both partial reactions. Here we show by electron paramagnetic resonance spectroscopy that binding of substrate L-arginine to neuronal NOS perturbs the heme cofactor binding pocket without directly interacting as a sixth axial heme ligand; heme iron is exclusively high spin. In contrast, binding of L-thiocitrulline, a NOS inhibitor, produces both high and low spin iron spectra; L-thiocitrulline sulfur is a sixth axial heme ligand in one, but not all, of the low spin forms. The high spin forms of the L-thiocitrulline NOS complex display a distortion in the opposite direction to that caused by L-arginine binding. The findings elucidate the binding interactions of L-arginine and L-thiocitrulline to neuronal NOS and demonstrate that each causes a unique perturbation to the heme cofactor pocket of NOS.
D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D002956:Citrulline / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics; D013890:Thiourea / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; C086715:thiocitrulline; D002956:Citrulline; D006418:Heme; D001120:Arginine; D019001:Nitric Oxide Synthase; D013890:Thiourea