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.
Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20 K, are reported, together with electron paramagnetic resonance spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the currently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling may be necessary for the QS state.
A direct relationship between the n C C stretching wavenumber and the orientations of the vinyl groups, as induced by specific protein interactions, is established for the superfamily of plant peroxidases. The present results help to rationalize the wavelength of the electronic absorption maxima of the overall spectrum of heme proteins and, in particular, of the Soret band. Moreover, inferences on the origin of the vinyl conformations in terms of the interactions with neighboring residues are drawn on the basis of the combined analysis of the x-ray structures, the amino acid sequence alignment and the spectroscopic data. On the basis of the distances between the C a and C b atoms of the vinyl groups and the surrounding atoms, the origin of the different vinyl orientations is ascribed to a variety of interactions, namely van der Waals contacts, weak p-p electron interactions and C -H· · ·O hydrogen bonds. The vinyl group orientations appear to depend on the concerted orientations of the distal helix B and proximal helix F axes as controlled by the H-bonds between the distal Arg, a heme propionyl group and a residue on the extended strand adjacent to proximal helix F. Finally, a possible mechanism explaining the alkaline configuration changes of cytochrome c peroxidase and the reorientation of a vinyl group upon fluoride complexation is given.
The preresonance Raman spectra, taken under various conditions, of crystal violet have been obtained using the exciting lines of an Ar+ laser. The analysis of the Raman data and of the IR and UV visible absorption spectra allowed us to determine the structure of the ion (propeller-like in shape with D3 symmetry), to characterize the unstable single protonated dye cation, and to propose a detailed vibrational assignment in terms of coupled vibrations of the substituted benzenes. In addition, the measurements of the Raman excitation profiles furnished information on the vibronic couplings and provided evidence for a close correlation in the resonance mechanisms between crystal violet and benzene and its monosubstituted derivatives.
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.
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