The nitric oxide (NO) produced by inducible nitric oxide synthase (NOS) 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 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.