The binding of a variety of ligands with Fe(III)-heme(+) ion, prosthetic group of heme proteins, has been studied in the gas phase by ESI-FT-ICR mass spectrometry. The ligands have been selected among substrate molecules of heme proteins (e.g., NO, nitroso compounds) or among model compounds acting for the functional groups that are present in the protein backbone (e.g., amines, thioethers, nitriles, ketones, amides, etc.). Both the kinetic and the thermodynamic features of the addition reactions are reported. Fe(III)-heme(+) ions react faster with lone pair donor ligands as the reaction becomes increasingly thermodynamically favored (higher heme cation basicity of the ligand, HCB, namely -DeltaG degrees for the ligand addition reaction). In turn HCBs correlate in general with the gas phase basicity toward the proton of the various ligands. A ligand addition equilibrium is established with weaker ligands, methanol, acetonitrile and acetone, yielding absolute HCB values, whereas ligand transfer equilibriums allowed a scale of relative (and absolute) HCBs to be constructed. NO displays exceptional binding properties towards Fe(III)-heme(+), unrelated to the low gas phase basicity toward the proton of this molecule, which is clearly the basis for the paramount role of heme proteins in NO binding and regulation.