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Flexibility is an important property of porphyrins, both natural and synthetic. We applied two synchrotron-based techniques, nuclear resonance vibrational spectroscopy and inelastic X-ray scattering, to quantify this property by measuring the bulk modulus of a protein active-site mimic [chloro(octaethylporphyrinato)iron(III)] and the resilience of the iron environment. Their values are 6.95 ± 0.24 GPa and 15.4 ± 0.5 N/m, respectively.

Nuclear resonance vibrational spectroscopy (NRVS) and Raman spectroscopy on (54)Fe- and (57)Fe-enriched cytochrome c (cyt c) identify multiple bands involving vibrations of the heme Fe. Comparison with predictions from Fe isotope shifts reveals that 70% of the NRVS signal in the 300-450 cm(-1) frequency range corresponds to vibrations resolved in Soret-enhanced Raman spectra. This frequency range dominates the "stiffness", an effective force constant determined by the Fe vibrational density of states (VDOS), which measures the strength of nearest-neighbor interactions with Fe. The stiffness of the low-spin Fe environment in both oxidation states of cyt c significantly exceeds that for the high-spin Fe in deoxymyoglobin, where the 200-300 cm(-1) frequency range dominates the VDOS. This situation is reflected in the shorter Fe-ligand bond lengths in the former with respect to the latter. The longer Fe-S(Met80) in oxidized cyt c with respect to reduced cyt c leads to a decrease in the stiffness of the iron environment upon oxidation. Comparison with NRVS measurements allows us to assess assignments for vibrational modes resolved in this region of the heme Raman spectrum. We consider the possibility that the 372 cm(-1) band in reduced cyt c involves the Fe-S(Met80) bond.