CO exchange of the oxyferrous complexes of endothelial nitric-oxide synthase oxygenase domain in the presence of 4-amino-tetrahydrobiopterin.
Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a 1-electron donor to the oxyferrous-heme complex. 4-Amino-tetrahydrobiopterin (4-amino-BH4) inhibits NO synthesis, although it has similar redox properties. We recently reported that 4-amino-BH4 is capable of electron transfer to Fe(II).O(2) in cryogenic single-turnover [J. Biol. Chem. 278 (2003) 48602]. We also suggested that BH4 serves as a proton donor to the Fe(II).O(2)(-) complex, and that 4-amino-BH4 cannot perform this second essential function. To corroborate these claims and to further characterize the intermediates observed after oxygenation of NOS in the presence of 4-amino-BH4, we added CO immediately after O(2) addition to the reduced oxygenase domain of endothelial NOS at -30 degrees C. This resulted in complete formation of a P450-type Fe(II).CO complex with either Arg or NG-hydroxy-L-arginine as the substrate. In the presence of 4-amino-BH2, which is redox-inactive, the same procedure yielded ferric heme with either substrate, without formation of any Fe(II).CO complex. We conclude: (i) O(2) binding to ferrous heme in the presence of 4-amino-BH2 is essentially irreversible; (ii) 4-amino-BH4 can reduce the oxyferrous complex; (iii) O(2)(-), rather than H(2)O(2) is the immediate product of uncoupled catalysis in the presence of 4-amino-BH4.
Marchal
Stéphane
S
INSERM U431, Département Biologie-Santé, Université Montpellier II, IFR 122, Montpellier, France.
Lange
Reinhard
R
Sørlie
Morten
M
Andersson
K Kristoffer
KK
Gorren
Antonius C F
AC
Mayer
Bernd
B
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry*; D002248:Carbon Monoxide / Q000737:chemistry*; D002417:Cattle; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052250:Nitric Oxide Synthase Type III; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary
D005296:Ferrous Compounds; D001708:Biopterins; D006418:Heme; D002248:Carbon Monoxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin; C034236:ferrous oxide