Resonance Raman studies of methemoglobin derivatives at room temperature and 77 K.

Raman spectra in preresonance with the Soret absorption band are reported for the following methemoglobin derivatives: cyanide, cyanate, thiocyanate, hydroxy-, azide, and fluoride methemoglobin at 285 K and 77 K. For the mixed-spin derivatives, Raman intensity is observed to shift from the high-spin marker band (approx. 1480 cm-1) to the low-spin marker band (approx. 1505 cm-1) upon cooling to 77 K. In addition, Raman spectra of cyanate methemoglobin were taken as a function of temperature, and the log of the intensity ratio I1480/I1505 was found to be a linear function of 1/T, indicating a thermally activated process. We interpret these results as observations of temperature-induced spin transitions. In the case of cyanate methemoglobin we find the enthalpy and entropy differences between the high-spin and low-spin states to be deltaH = 600 +/- 40 cal x mol-1 and deltaS = 4.7 +/- 0.7 cal x mol-1 x K-1. The high-spin to low-spin ratio for cyanate methemoglobin determined by our experiment disagrees with the value reported for magnetic susceptibility measurements.

Resonance Raman investigations of chloroperoxidase, horseradish peroxidase, and cytochrome c using Soret band laser excitation.

Resonance Raman spectra of the heme protein chloroperoxidase in its native and reduced forms and complexed with various small ions are obtained by using laser excitation in the Soret region (350-450 nm). Additionally, Raman spectra of horseradish peroxidase, cytochrome P-450cam, and cytochrome c, taken with Soret excitation, are presented and discussed. The data support previous findings that indicate a strong analogy between the active site environments of chloroperoxidase and cytochrome P-450cam. The Raman spectra of native chloroperoxidase are found to be sensitive to temperature and imply that a high leads to low spin transition of the heme iron atom takes place as the temperature is lowered. Unusual peak positions are also found for native and reduced chloroperoxidase and indicate a weakening of porphyrin ring bond strengths due to the presence of a strongly electron-donating axial ligand. Enormous selective enhancements of vibrational modes at 1360 and 674 cm-1 are also observed in some low-spin ferrous forms of the enzyme. These vibrational frequencies are assigned to primary normal modes of expansion of the prophyrin macrocycle upon electronic excitation.