QUANTITATIVE STRUCTURAL COMPARISONS OF HEME PROTEIN CRYSTALS AND SOLUTIONS USING RESONANCE RAMAN-SPECTROSCOPY

Resonance Raman difference spectra have been used to compare crystal a nd solution samples of metmyoglobin (metMb), deoxymyoglobin (deoxyMb), and cytochrome P450. At pH 6.0, the frequency shifts of the heme core size sensitive bands nu2, nu3, and nu4 were determined to be less tha n 0.3, 1.0, and 0.3 cm-1, respectively, for metMb and to be less than 1.0, 1.0, and 0.3 cm-1, respectively, for deoxyMb. This shows that the heme core size differences between the crystal and solution conformat ions are less than 0.002 angstrom for metMb and less than 0.003 angstr om for deoxyMb. These results disagree with a recent extended X-ray ab sorption fine structure study [Zhang, K., Chance, B., Reddy, K. S., Ay ene, I., Stern, E. A., & Bunker, G. (1991) Biochemistry 30, 9116-9120] which claims that a 0.05-angstrom difference exists in the average ir on-ligand distance between the crystalline and solution forms of metMb at pH 6.5. At pH 8.5, metMb solution samples change gradually from a predominantly high-spin to a predominantly low-spin species as the amm onium sulfate concentration is increased to the level found in the cry stal mother liquor. No Raman frequency shifts are found between the cr ystal and solution forms of metMb at pH 8.5 when the ammonium sulfate concentrations are equal. On the other hand, for deoxyMb, we find a si gnificant alteration in the 220/240-cm-1 line shape and relative inten sities, suggesting that some histidine-heme perturbation takes place u pon crystallization. A continuous-wave photolysis experiment shows tha t when the MbCO crystal is photolyzed, there is a slow (tau is similar to 100 s) increase of.the photolyzed population with time. We suggest that this is due to CO moving into the protein matrix of the crystall ized protein, leading to long-lived deoxy states, similar to those fou nd in frozen MbCO samples near the glass transition of the solvent.