Genealogy of mammalian cysteine proteinase inhibitors. Common evolutionary origin of stefins, cystatins and kininogens

Resonance Raman spectra are reported for native horseradish peroxidase (HRP) and cytochrome c peroxidase (CCP) at 290, 77 and 9 K, using 406.7 nm excitation, in resonance with the Soret electronic transition. The spectra reveal temperature-dependent equilibria involving changes in coordination or spin state. At 290 K and pH 6.5, CCP contains a mixture of 5- and 6-coordinate high-spin Fe^III heme while at 9 K the equilibrium is shifted entirely to the 6-coordinate species. The spectra indicate weak binding of H₂O to the heme Pe, consistent with the long distance, 2.4 Å, seen in the crystal structure. At 290 K HRP also contains a mixture of high-spin Fe^III hemes with the 5-coordinate form predominant. At low temperature, a small 6-coordinate high-spin component remains but the 5-coordinate high-spin spectrum is replaced by another which is characteristic either of 6-coordinate low-spin or 5-coordinate intermediate spin heme. The latter species is definitely indicated by previous EPR studies at low temperature. This behavior implies that, in contrast to CCP, the distal coordination site of HRP is only partially occupied by H₂O at any temperature and that lowering the temperature significantly weakens the Fe-proximal imidazole bond. Consistent with this inference, the 77 K spectrum of reduced HRP shows an appreciable fraction of molecules having an Fe-imidazole stretching frequency of 222 cm^?1, a value indicating weakened H-bonding of the proximal imidazole.

Resonance Roman spectroscopy Horseradish peroxidase Cytochrome c peroxidase Coordination equilibrium