Stoichiometric arginine binding in the oxygenase domain of inducible nitric oxide synthase requires a single molecule of tetrahydrobiopterin per dimer.
In addition to its catalytic roles, the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (H4B) is required for substrate binding and for stabilization of the dimeric structure. We expressed and purified the core of the iNOS oxygenase domain consisting of residues 75-500 (CODiNOS) in the presence (H4B+) and absence (H4B-) of this cofactor. Both forms bound stoichiometric amounts of heme (>0.9 heme per protein subunit). H4B- CODiNOS was unable to bind arginine, gave an unstable ferrous carbonyl adduct, and was a mixture of monomer and dimer. H4B+ CODiNOS bound arginine, gave a stable ferrous carbonyl adduct, and was exclusively dimeric. The H4B cofactor content of this species was only one per dimer yet this was sufficient to form two competent arginine binding sites as determined by optical stoichiometric titrations.
Rafferty
S P
SP
Office of Scientific Directors, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland, 20892-1886, USA. srafferty@trentu.ca
Boyington
J C
JC
Kulansky
R
R
Sun
P D
PD
Malech
H L
HL
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D001708:Biopterins / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D002248:Carbon Monoxide / Q000494:pharmacology; D002850:Chromatography, Gel; D017124:Conserved Sequence; D019281:Dimerization; D004227:Dithionite / Q000494:pharmacology; D005296:Ferrous Compounds / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D051379:Mice; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D017186:Titrimetry
D005296:Ferrous Compounds; D011994:Recombinant Proteins; D004227:Dithionite; D001708:Biopterins; D006418:Heme; C017226:7,8-dihydrobiopterin; D002248:Carbon Monoxide; D001120:Arginine; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C003402:sapropterin