Abstract
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.
Highlights
Nitric-oxide synthase (NOS,1 EC 1.14.13.39) converts L-arginine into L-citrulline and NO in two consecutive reaction cycles with intermediate formation of NG-hydroxy-L-arginine (NHA)
We studied the reaction of reduced endothelial nitric-oxide synthase (NOS) oxygenase domain with O2 in the presence of 4-amino-BH4 at ؊30 °C by optical and electron paramagnetic resonance (EPR) spectroscopy
Effect of 4-Amino-BH4 on NO Synthesis—Previous studies have shown that 4-amino-BH4 is a strong BH4-competitive, reversible inhibitor of NO synthesis, measured as conversion of radiolabeled arginine and NHA to citrulline [16, 17, 19]
Summary
Nitric-oxide synthase (NOS, EC 1.14.13.39) converts L-arginine into L-citrulline and NO in two consecutive reaction cycles with intermediate formation of NG-hydroxy-L-arginine (NHA) (see Refs. 1–3 for recent reviews). BH4 performs the same function in the second cycle as well [9, 13, 14], little or no BH3 radical is observed (8 –10), probably because the different electronic requirements of the second cycle allow rapid regeneration of BH4 [4, 13]. As yet it is unclear whether BH4 participates in later reaction steps such as proton donation or stabilization of the putative ferryl1⁄7oxy complex [4]. Erythro-1Ј,2Ј-dihydroxypropyl)pteridine); BH3 and 4-amino-BH3, the one-electron oxidized radical compounds corresponding to BH4 and 4-amino-BH4, respectively; NHA, NG-hydroxy-L-arginine; CaM, calmodulin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; SOD, superoxide dismutase; CAT, catalase; EPR, electron paramagnetic resonance
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