Probing Heme Coordination States of Inducible Nitric Oxide Synthase with a Re(I)(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 O_2. The Soret peak in the spectrum of the iNOS heme domain (iNOS_(oxy)) shifts from 423 to 390 nm upon addition of a sensitizer-wire, [Re^I-imidazole-(CH_2)_8-nitroarginine]^+, or [ReC_8argNO_2]^+, owing to partial displacement of the water ligand in the active site. From analysis of competitive binding experiments with imidazole, the dissociation constant (K_d) for [ReC_8argNO_2]^+−iNOS_(oxy) was determined to be 3.0 ± 0.1 μM, confirming that the sensitizer-wire binds with higher affinity than both L-arginine (K_d = 22 ± 5 μM) and imidazole (K_d = 14 ± 3 μM). Laser excitation (355 nm) of [ReC_8argNO_2]^+−iNOS_(oxy) triggers electron transfer to the active site of the enzyme, producing a ferroheme in less than ∼1 μs.