Structural Characterization of Cytochrome c- under Peroxidase by Resonance Raman Scattering.

Resonance Raman Scattering studies are reported on freshly prepared ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c- peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin independent of buffer over the pH range of 4.3-7 as determined by well established Raman marker lines. The "aged" enzyme displays a mixture of spin and coordination states but may be stabilized in the penta-coordinate high spin form by the presence of phosphate (0.2M). These results can be accounted for by considering the size of the channel (~ 6angstrom wide by 11angstrom long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated by the channel resulting in a proposed stabilization of the distal heme pocket. The ferrous enzyme in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic peroxidase spectral features - a high frequency iron-histidine stretching mode (247 cm sup (-1)), high frequency Fe-CO stretching mode 537 cm sup (-1)), and a low frequency C-O stretching mode (1922 cm sup (-1)). At alkaline pH the spectral features change and become quite similar to those of hemoglobin and myoglobin with the corresponding modes located at 227, 510, and 1948 cm sup (-1), respectively. We attribute the acid/alkaline transition in the ferrous forms of the enzyme to a structural change on the proximal side of the heme culminating in a change in steric interactions between the proximal histidine and the heme or changes in the hydrogen bonding network involving the proximal histidine. The new data report here reconcile the many apparent conflicts reported in the past.