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

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 H₂O-D₂O 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 ν_Fe-CO and ν_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.