Spectroscopic evidence for a conformational transition in horseradish peroxidase at very low pH.

Resonance Raman (RR), electronic absorption, and circular dichroism (CD) spectroscopies of the ferric, ferrous, and ferrous-CO forms of horseradish peroxidase (HRP-C) at pH 3.1 are reported. The CD spectra in the UV region show only a small decrease in the alpha-helical content upon pH lowering, whereas dramatic changes are observed in the Soret region. The final form of ferric HRP-C is 5-coordinate high-spin heme whose histidine ligand is replaced by a water ligand with a polar character. The electronic and CD spectra show the presence of an intermediate form with a 6-coordinate heme. Therefore, the cleavage of the proximal Fe-imidazole bond is preceded by the binding of a distal water molecule. For the ferrous form of HRP-C, the pH-dependence of the absorption spectra revealed only the native form in the range pH 5-7 and an unfolded form with a Soret maximum at 383 nm at pH 3.1. An intermediate state, characterized by a Soret maximum at 424 nm, was observed only in a transient way, within a few milliseconds. A metastable and a final species are observed also for the ferrous-CO complex at pH 3.1, as proved by isosbestic points in the electronic absorption spectra. The two forms show different RR nu(Fe-C) and IR nu(CO) modes. The metastable form corresponds to a heme where histidine is replaced by water. The final form is due to the displacement of the water ligand by the proximal histidine. We propose a kinetic model to account for our results at pH 3.1 for the ferric, ferrous, and ferrous-CO forms.

Resonance Raman studies of the heme active site of the homodimeric myoglobin from Nassa mutabilis: a peculiar case.

A spectroscopic investigation by resonance Raman has been carried out at pH 7.0 in 0.1 M phosphate buffer on the cooperative homodimeric myoglobin from Nassa mutabilis. The study has been performed on the unligated ferrous form, as well as on the ligated species MbO2 and MbC, and on the ferric form met-Mb. Two v(C = C) vinyl stretching modes have been observed in all the investigated forms, reflecting different degrees of vinyl conjugation with the porphyrin ring, as a consequence of a strongly asymmetric environment for the two side groups of the heme. Furthermore, the ferric form displays a hexacoordinate low-spin heme, which suggests the presence of an endogenous ligand bound to the Fe atom. The frequency of the v(Fe-Im) stretching mode of Mb from Nassa mutabilis shifts down by 4 cm-1 as compared with that of horse heart myoglobin, reflecting a protein-induced proximal strain as a result of heme-heme interaction due to the close proximity of the two hemes in the dimer. The lower frequency of the v(Fe-Im) stretching mode agrees well with the lower affinity for oxygen binding found for Nassa mutabilis Mb and with the slight heme core expansion with respect to horse heart Mb, suggesting a critical role for the Fe-His bond on the heme's function and structure.

Alteration of the proximal bond energy in the unliganded form of the homodimeric myoglobin from Nassa mutabilis. Kinetic and spectroscopic evidence.

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.0. Protonation of the proximal imidazole at low pH (less than or equal to 3.0) and the consequent cleavage of the HisF8NE2-Fe proximal bond brings about a approximately 20-fold increase of the second-order rate constant for CO binding. This process displays a pKa = 4.0 +/- 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2-Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH-rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828-4830), can be described only by assuming a concerted proton-linked transition with n = 1.8 +/- 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.

Resonance Raman investigation of ferric iron in horseradish peroxidase and its aromatic donor complexes at room and low temperatures.

Resonance Raman (RR) spectra of the acidic form of FeIII horseradish peroxidase (HRP) were obtained at room and low temperatures using B- and Q-band excitation. At 296 K, HRP exhibits two sets of porphyrin skeletal stretching frequencies which are attributed to a thermal mixture of 5- and 6-coordinate high-spin FeIII states. When the temperature is lowered, the observed bands shift to higher frequencies, and these are assigned to intermediate- and low-spin states. Addition of 40% glycerol has no effect on the spectra at 296 K, but at 20 K, all four frequency sets are observed corresponding to the two forms observed at room and low temperature in the absence of glycerol. The 296 K RR spectrum of the HRP-hydroquinone complex is similar to that of free HRP, but conversion to the intermediate- and low-spin states is complete at a higher temperature than in the free enzyme. Addition of benzohydroxamic acid (BHA) to HRP shifts the RR frequencies to those corresponding to a 6-coordinate high-spin species at both room and low temperature. Two upsilon (C = C) stretching modes are observed for HRP and its donor complexes, indicating that the vinyl groups are inequivalent. On BHA binding, one of the vinyl modes and upsilon 37 (Eu) are enhanced, suggesting symmetry lowering of the heme site.

Effects of temperature and glycerol on the resonance Raman spectra of cytochrome c peroxidase and selected mutants.

The high-frequency resonance Raman spectra of FeIII yeast native cytochrome c peroxidase (CCP) and five of its mutants [CCP(MI), Phe-51, Leu-48, Lys-48, Asn-235, and Phe-191] were recorded in phosphate buffer, pH 7.0, and in glycerol/phosphate mixtures at 295 and 10 K. Glycerol induces heme coordination changes in some of the CCP mutants at room temperature. It apparently weakens the binding of the Fe atom to ligands in the distal heme cavity and drives the heme toward the 5-coordinate, high-spin state. At 10 K, native CCP and all the mutants (except Phe-51 which remains 6-coordinate, high-spin) show various distributions of spin and coordination states which differ from those observed at 295 K. Upon cooling in phosphate buffer, pH 7, and to a much lesser extent in 66% glycerol/phosphate, an internal strong-field ligand is coordinated to the Fe. A likely candidate is H2O-595, which could become a strong-field ligand on H-bonding and/or proton transfer to H2O-648, and/or the distal His-52. However, distal His-52 itself cannot be ruled out as the coordinating ligand considering that the Phe-51 mutant, which binds H2O-595 at room temperature, does not show a large 6-coordinate, low-spin component at 10 K like the other mutants. These results clearly indicate that the Fe coordination in CCP and its mutants is sensitive to both temperature and solvent composition.