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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.

SUBMITTER: Sabat J

PROVIDER: S-EPMC2882098 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Characterization of the proximal ligand in the P420 form of inducible nitric oxide synthase.

Sabat Joseph J Stuehr Dennis J DJ Yeh Syun-Ru SR Rousseau Denis L DL

Journal of the American Chemical Society 20090901 34

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) specie ...[more]

PMID: 19658411

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Project description:UnlabelledInducible nitric oxide synthase (iNOS) activity increases in acute and chronic inflammatory lung diseases. Imaging iNOS expression may be useful as an inflammation biomarker for monitoring lung disease activity. We developed a novel tracer for PET that binds to iNOS in vivo, (18)F-NOS. In this study, we tested whether (18)F-NOS could quantify iNOS expression from endotoxin-induced lung inflammation in healthy volunteers.MethodsHealthy volunteers were screened to exclude cardiopulmonary disease. Qualifying volunteers underwent a baseline, 1-h dynamic (18)F-NOS PET/CT scan. Endotoxin (4 ng/kg) was then instilled bronchoscopically in the right middle lobe. (18)F-NOS imaging was performed again approximately 16 h after endotoxin instillation. Radiolabeled metabolites were determined from blood samples. Cells recovered by bronchoalveolar lavage (BAL) after imaging were stained immunohistochemically for iNOS. (18)F-NOS uptake was quantified as the distribution volume ratio (DVR) determined by Logan plot graphical analysis in volumes of interest placed over the area of endotoxin instillation and in an equivalent lung region on the left. The mean Hounsfield units (HUs) were also computed using the same volumes of interest to measure density changes.ResultsSeven healthy volunteers with normal pulmonary function completed the study with evaluable data. The DVR increased by approximately 30%, from a baseline mean of 0.42 ± 0.07 to 0.54 ± 0.12, and the mean HUs by 11% after endotoxin in 6 volunteers who had positive iNOS staining in BAL cells. The DVR did not change in the left lung after endotoxin. In 1 volunteer with low-level iNOS staining in BAL cells, the mean HUs increased by 7% without an increase in DVR. Metabolism was rapid, with approximately 50% of the parent compound at 5 min and 17% at 60 min after injection.Conclusion(18)F-NOS can be used to image iNOS activity in acute lung inflammation in humans and may be a useful PET tracer for imaging iNOS expression in inflammatory lung disease.

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Characterization of the proximal ligand in the P420 form of inducible nitric oxide synthase.

Publications

Characterization of the proximal ligand in the P420 form of inducible nitric oxide synthase.

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