Heme proteins: Structure and function
Abstract
Resonance Raman (RR) spectroscopy has been employed to investigate the structure of the cyanide adducts of horseradish peroxidase (HRP) in the pH range 5.5 to 12.5. Evidence for the binding of cyanide in two forms is observed. In the first conformer the $\nu\lbrack$Fe-CN) stretching mode occurs at higher frequency than the $\delta\lbrack$Fe-C-N) bending mode, behavior characteristic of an essentially linear Fe-C-N linkage. In the second conformer this ordering is reversed, indicative of a bent Fe-C-N linkage. Furthermore, the axial mode vibrations of the Fe-C-N linkage of both conformers were found to shift upon raising the pH, consistent with deprotonation of a distal histidine. Investigation of cyanide bound beef liver catalase (BLC) and Aspergillus niger catalase (ANC) in the pH range 4.9-11.5 revealed a similar spectral behavior which is also indicative of the presence of linear and bent conformers. The two conformers persist in the pH range 4.9-11.5. At high pH, the $\nu\lbrack$Fe-CN) mode of the linear conformer of BLC-CN shifts to lower frequency while that of the bent conformer remains unchanged. The propensity for cyanide to bind to these proteins in two conformations and the observed pH dependent shifts are attributable to distal- and proximal-side interactions that modulate the binding geometry of the cyanide ligand. The reactive intermediates of HRP and mesoheme-substituted HRP (meso-HRP) have been investigated by RR spectroscopy. The high frequency RR spectra of HRP compound I reveal a behavior that is consistent with the presence of a $\pi$-cation radical having an A$\sb{\rm 2u}$ ground state electronic configuration. However, meso-HRP compound I displayed a behavior that is characteristic of an A$\sb{\rm 1u}$ configuration. This alteration in configuration is reasonably attributable to the change in the nature of the heme peripheral substituents. Investigation of the low frequency spectra of meso-HRP compound I revealed an isotope-sensitive mode at 794 cm$\sp{-1}$ that has been assigned to the $\nu\lbrack$Fe=O) stretching vibration.
This paper has been withdrawn.