Absorption and resonance Raman spectra using Soret excitation of alkaline metmyoglobin (metMb), methemoglobin (metHb), and horseradish peroxidase (HRP) were obtained at room and low temperature. At 298 K both metMb and metHb exhibit two isotope-sensitive bands assigned to high- and low-spin nu(Fe-OH) stretching modes, respectively, which are correlated with the spin-state population. The low-spin stretch occurs 60 cm-1 to higher energy than the corresponding high-spin vibration. When the temperature is lowered, only the low-spin species is observed. HRP exhibits at both 298 and 20 K only the low-spin nu(Fe-OH) stretching mode, which occurs 50 cm-1 to lower energy than the corresponding modes observed in the globins. This is explained in the context of a strong hydrogen bond between the hydroxyl ligand and the distal His42 and/or Arg38. Lowering temperature causes in all of the examined proteins a strengthening of the Fe-OH bond and a contraction of the core of about 0.01 A, as determined by the upshifting of the low-spin nu(Fe-OH) stretching mode and the core size marker bands. Both effects are ascribed to an increase of the packing forces.

Resonance Raman spectra are reported for recombinant horseradish peroxidase C (HRP-C*) and three protein variants prepared by in vitro refolding after Escherichia coli expression. The spectra of their FeII and FeIII forms and of their complexes with benzohydroxamic acid (BHA) were recorded at neutral pH. The residues mutated were on the distal [Phe41-->Trp or Val (F41W, F41V) and Arg38-->Lys (R38K)] side of the heme. The spectra give information on the spin and ligation states via the frequencies of the core size marker bands. No detectable modification in the enzyme structure or in the heme group has been observed in the wild-type recombinant HRP-C*. The FeIII forms of both the recombinant and the plant proteins show the coexistence of a 5-(5-cHS) and a 6-coordinate high-spin (6-cHS) heme, characterized by the anomalous frequencies of certain bands, namely, v3 and v10, which we attribute to a different degree of distortion of the heme planarity with respect to other heme proteins and model compounds, resulting from external forces such as steric contacts within the protein. This effect is partially relieved upon complexation with BHA or as a result of mutation. F41W and F41V are characterized by an increase in a 6-cHS form at the expense of the 5-cHS species, and the R38K by an increase in both the 6-c high-(HS) and low-spin (LS) hemes. The 6-cHS and -LS species are characterized by normal core size marker band frequencies. The FeII-His RR band is at 243 cm-1 in HRP-C*, the high frequency value being due to hydrogen-bonding interactions between the proximal His170 N delta and the carboxylate acceptor group on Asp247. Mutation at position 38 causes a downshift of 3 cm-1 in the v(Fe-Im) stretching mode, suggesting a weakening of the Fe-Im bond strength. By comparing the results obtained with HRP-C* mutants with those previously reported for the corresponding cytochrome c peroxidase (CCP) mutants, it appears that the distal heme pocket architecture is significantly different in the two peroxidases, although the hydrogen-bonding network coupling the distal and the proximal sides of the heme appears to be conserved. Mutations on the distal side dramatically affect the capability of the protein to bind BHA. F41W and R38K mutants do not bind the substrate, whereas the F41V variant shows a 2-fold increase in affinity.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

This paper analyzes some critical security issues in the family of IEEE 802.16 standard that has not been addressed so far. In particular two of the key features of the standard, the dynamic resources allocation and the mesh mode revealed to be vulnerable to attacks that represent serious threats to the robustness and privacy of the communications. In the first case the attacker is able to reduce bandwidth assigned to its neighbors, with the aim of obtaining more resources for himself; in the second case, we observed that there might be no real privacy in communications between two nodes of the mesh network. These vulnerabilities are still present even after the latest amendment to the standard, IEEE 802.16e that solved some previously addressed security flaws.