The porphyrinic metal-organic framework (MOF) PCN-224 is metalated with FeII to yield a 4-coordinate ferrous heme-containing compound. The heme center binds O-2 at -78 degrees C to give a 5-coordinate heme-O-2 complex. For the first time, this elusive species is structurally characterized, revealing an Fe-III center coordinated to superoxide via an end-on, eta(1) linkage. Mossbauer spectroscopy supports the structural observations and indicates the presence of a low-spin electronic configuration for FeIII. Finally, variable-temperature O-2 adsorption data enable quantification of the Fe-O-2 interaction, providing a binding enthalpy of -34(4) kJ/mol. This value is nearly half of that observed for comparable 6-coordinate, imidazole-bound heme-O-2 complexes, a difference that further illustrates the importance of axial ligands in biological heme-mediated O-2 transport and storage. These results demonstrate the ability of a MOF, by virtue of its rigid solid-state structure, to enable isolation and thorough characterization of a species that can only be observed transiently in molecular form.