| pubmed-article:14514694 | pubmed:abstractText | Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a one-electron donor to the oxyferrous.heme complex. 4-Aminotetrahydrobiopterin (4-amino-BH4) is a potent inhibitor of NO synthesis, although it mimics all allosteric and structural effects of BH4 and exhibits comparable redox properties. We studied the reaction of reduced endothelial NOS oxygenase domain with O2 in the presence of 4-amino-BH4 at -30 degrees C by optical and electron paramagnetic resonance (EPR) spectroscopy. With Arg as the substrate, we observed a trihydropteridine radical with a corresponding heme species that was oxyferrous, with a Soret maximum at 428 nm and no EPR signal. With NG-hydroxy-l-arginine (NHA) no pterin radical appeared, whereas an axial ferrous heme.NO complex was formed. The corresponding optical spectra, with Soret bands at 417/423 nm, suggest that the proximal sulfur ligand is protonated. Accordingly, 4-amino-BH4 serves as a one-electron donor to Fe(II).O2 with both Arg and NHA, but the reaction cycle cannot be completed with either substrate. We propose that protonation of Fe(II)O2- is inhibited in the presence of 4-amino-BH4. With Arg, dissociation of O2- and binding of O2 yields Fe(II).O2 and a pteridine radical; with NHA, reaction of the substrate with heme-bound O2- eventually yields Fe(II).NO and reduced 4-amino-BH4. These results suggest that BH4 donates a proton to Fe(II).O2- during catalysis and that inhibition by 4-amino-BH4 may be due to its inability to support this essential protonation step. | lld:pubmed |
