Core expansion and electronic structure of the porphyrin in the neutral pH form of copper cytochrome c

The pH-dependent resonance Raman and absorption spectra of copper cytochrome c are examined. Large shifts in the Raman core-size marker lines are observed in the neutral pH form of copper cytochrome c relative to the protein at pH extremes (pH 2 and 13). The 5-coordinate copper-porphyrin complexes exhibit similar, but somewhat smaller, shifts in the core-size marker lines. The Raman shifts represent a significant expansion (0.03 angstrom) of the porphinato core in the neutral form. Further, analysis of the pi arrow right pi * absorption spectral changes using the four-orbital model indicates a substantial destabilization of the top-filled a sub(2u)( pi ) orbital if the neutral pH form of the protein has 5-coordinate copper. The results is a red shift of all pi arrow right pi * bands, a decrease in the separation of the alpha -band and Soret band, and a reversal in the alpha - and beta -band absorbance ratio from similar to 1.4 to similar to 0.8. Iterative extended Hueckel molecular orbital (MO) calculations for model 4-, 5-, and 6-coordinate copper porphyrins show considerable destabilization of the a sub(2u) orbital caused by addition of a pi -donating axial ligand. Thus, the calculations support previous interpretations of spectral changes based on addition of an axial fifth ligand in the copper-modified heme proteins and copper porphyrins in coordinating solvents.