Resonance Raman spectroscopy of cytochrome c peroxidase single crystals on a variable-temperature microscope stage.

Good quality resonance Raman (RR) spectra have been obtained for cytochrome c peroxidase single crystals (0.2 x 0.5 x 1 mm) lying on their 110 faces on a microscope stage. Crystal orientation and polarization effects are observed which differentiate the RR bands on the basis of the symmetries of the porphyrin vibrational modes. The measured depolarization ratios are accurately calibrated for isolated bands of both totally symmetric and non totally symmetric modes by using a model of D4h chromophores in an oriented gas using the crystal structure atomic coordinates. The calculations indicate that the electronic transition moments are approximately along the lines connecting the methine bridges, suggesting an electronic steering effect of the vinyl groups. Deviations are observed for bands associated with the porphyrin v10 and the vinyl C = C stretching modes, which may be due to their near-resonant interaction. The band frequencies correspond to those of a five-coordinate high-spin FeIII heme, as previously observed in solution, consistent with the X-ray structure showing the Fe atom to be out of the heme plane on the proximal side with a distal water molecule located at a nonbonded distance, 2.4 A. The temperature dependence of the RR spectrum was determined with a Joule-Thompson cryostat on crystals sealed in glass capillaries. As the temperature is lowered, the spectrum converts to one characteristic of a low-spin FeIII heme. The conversion, which is readily reversible, is quite gradual. It is detectable at -50 degrees C but is incomplete even at -190 degrees C. A temperature effect on the protein structure is proposed which permits the Fe atom to approach the heme plane and bind the distal water molecule, or the distal histidine.