Abstract

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.

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