title: Characterization of the proximal ligand in the P420 form of inducible nitric oxide synthase.
abstract: The nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) up-regulates the expression of heme oxygenase (HO), which in turn produces carbon monoxide (CO) that down-regulates iNOS activity by reducing its expression level or by inhibiting its activity by converting it to an inactive P420 form (iNOS(P420)). Accordingly, CO has been considered as a potentially important attenuator of inflammation. Despite its importance, the nature of the proximal heme ligand of the iNOS(P420) species remains elusive. Here we show that the 221 cm(-1) mode of the photoproduct of iNOS(P420) does not exhibit any H(2)O-D(2)O solvent isotope shift such as that found in the iron-histidine stretching mode of myoglobin, indicating that the proximal ligand of iNOS(P420) is not a histidine. The nu(Fe-CO) and nu(C-O) data reveal that the proximal heme ligand of iNOS(P420) is consistent with a protonated thiol instead of a thiolate anion. Furthermore, the optical absorption properties of iNOS(P420) are similar to those of a neutral thiol-heme model complex but not myoglobin. Together the data support the scenario that iNOS(P420) is inactivated by protonation of the native proximal thiolate ligand to a neutral thiol, instead of by ligand switching to a histidine, as prior studies have suggested.
authors: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors: Joseph
authors: J
authors: Sabat
authors: Dennis J
authors: DJ
authors: Stuehr
authors: Syun-Ru
authors: SR
authors: Yeh
authors: Denis L
authors: DL
authors: Rousseau
mesh_terms: D000042:Absorption; D000818:Animals; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006427:Hemin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D051379:Mice; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons; D013059:Spectrum Analysis, Raman; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013997:Time Factors
chemical_list: D007527:Isoenzymes; D008024:Ligands; D009211:Myoglobin; D011522:Protons; D013438:Sulfhydryl Compounds; C032548:hemin dimethyl ester; D006418:Heme; D006427:Hemin; D002248:Carbon Monoxide; D010105:Oxygenases; D052247:Nitric Oxide Synthase Type II