Probing heme coordination states of inducible nitric oxide synthase with a ReI(imidazole-alkyl-nitroarginine) sensitizer-wire.
Mammalian inducible nitric oxide synthase (iNOS) catalyzes the production of l-citrulline and nitric oxide (NO) from L-arginine and O2. The Soret peak in the spectrum of the iNOS heme domain (iNOSoxy) shifts from 423 to 390 nm upon addition of a sensitizer-wire, [ReI-imidazole-(CH2)8-nitroarginine]+, or [ReC8argNO2]+, owing to partial displacement of the water ligand in the active site. From analysis of competitive binding experiments with imidazole, the dissociation constant (Kd) for [ReC8argNO2]+-iNOSoxy was determined to be 3.0+/-0.1 microM, confirming that the sensitizer-wire binds with higher affinity than both L-arginine (Kd=22+/-5 microM) and imidazole (Kd=14+/-3 microM). Laser excitation (355 nm) of [ReC8argNO2]+-iNOSoxy triggers electron transfer to the active site of the enzyme, producing a ferroheme in less than approximately 1 micros.
Le Nguyen
Yen Hoang
YH
Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA.
Winkler
Jay R
JR
Gray
Harry B
HB
D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry*; D008958:Models, Molecular; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis / Q000737:chemistry*; D019335:Nitroarginine / Q000031:analogs & derivatives*; D009942:Organometallic Compounds; D013379:Substrate Specificity
D015335:Molecular Probes; D009942:Organometallic Compounds; C521571:Re-imidazole-(CH2)8-nitroarginine; D019335:Nitroarginine; D006418:Heme; D052247:Nitric Oxide Synthase Type II