Active site topologies and cofactor-mediated conformational changes of nitric-oxide synthases.
The active site topologies of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric-oxide synthases heterologously expressed in Escherichia coli have been examined using three aryldiazene (Ar-N=NH) probes. The topological information derives from (a) the rate and extent of aryl-iron complex formation in the presence and absence of tetrahydrobiopterin (H4B), Ca2+-dependent calmodulin (CaM), and L-arginine, and (b) the N-phenylprotoporphyrin IX regioisomer ratios obtained upon migration of the phenyl of the phenyl-iron complex to the heme nitrogen atoms. The N-phenylprotoporphyrin ratios indicate that the three NOS isoforms have related active site topologies with unencumbered space above all four pyrrole rings but particularly above pyrrole ring D. H4B binds directly above the heme pyrrole ring D or causes a conformational change that constricts that region, because H4B markedly decreases phenyl migration to pyrrole ring D. Small CaM-dependent changes in the nNOS N-phenylporphyrin isomer pattern are consistent with a conformational link between the CaM and heme sites in this protein. The ceiling height directly above the heme iron atom differs among the isoforms and is lower than in the P450 enzymes because only nNOS and iNOS react with 2-naphthyldiazene, and none of the isoforms reacts with p-biphenyldiazene. L-Arg blocks access to the heme iron atom in all three NOS isoforms and nearly suppresses the phenyldiazene reaction. The data indicate that topological differences, including differences in the size of the active site, are superimposed on the structural similarities among the NOS active sites.
Gerber
N C
NC
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Rodriguez-Crespo
I
I
Nishida
C R
CR
Ortiz de Montellano
P R
PR
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry; D002417:Cattle; D007094:Imides / Q000737:chemistry; D007097:Imines / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000648:ultrastructure*; D011487:Protein Conformation; D051381:Rats
D002147:Calmodulin; D007094:Imides; D007097:Imines; D001708:Biopterins; C011981:phenyldiazene; D001120:Arginine; D007501:Iron; D019001:Nitric Oxide Synthase; C003402:sapropterin; C038867:diazene