Resonance Raman spectra of native and mesoheme-reconstituted horseradish peroxidase and their catalytic intermediates.

Resonance Raman studies of native and mesoheme-reconstituted horseradish peroxidase and their catalytic intermediates, known as Compounds I and II, have been conducted using both near UV ( approximately 350 nm) and visible (406.7 nm) excitation. Careful power studies indicate that the authentic Compound I spectra are obtainable using near UV excitation, but that use of visible excitation results in contamination of the Compound I spectrum with the spectrum of a Compound II-like photoproduct. Using H218O2, the nu(Fe=O) stretching modes for both systems are unambiguously identified, for the first time, at approximately 790 cm-1. The authentic Compound I spectra are indicative of an 2A1u-like ground state for both the native and the mesoheme-reconstituted proteins. Finally, the possible biological implications of such information are briefly discussed.

Resonance Raman investigation of cyanide ligated beef liver and Aspergillus niger catalases.

Resonance Raman spectroscopy has been used to investigate the properties of cyanide-bound beef liver catalase (BLC) and Aspergillus niger catalase (ANC) in the pH range 4.9-11.5. Evidence has been obtained for the binding of cyanide to both BLC and ANC in two binding geometries. The first conformer, exhibiting the nu[Fe-CN] stretching mode at a higher frequency than the delta[Fe-C-N] bending mode, exists as an essentially linear Fe-C-N linkage. For both BLC-CN and ANC-CN, the nu[Fe-CN] and delta[Fe-C-N] frequencies of this conformer were practically identical and observed at approximately 434 and approximately 413 cm-1, respectively. The second conformer exhibits a nu[Fe-CN] mode at lower frequency than the delta[Fe-C-N] mode, and is thus characteristic of a bent Fe-C-N linkage. The nu[Fe-CN] and delta[Fe-C-N] modes were identified at 349 and 445 cm-1, respectively, for BLC-CN, and at 350 and 456 cm-1, respectively, for ANC-CN. The two conformers persist in the pH range 4.9-11.5. Furthermore, upon raising the pH to 11.5, the nu[Fe-CN] mode of the linear conformer of BLC-CN downshifts to 429 cm-1 while that of the bent conformer remains unchanged. The observed pH-dependent shift is attributed to the deprotonation of a distal-side amino acid residue, probably a distal histidine. The Fe-C-N axial vibrations of the two conformers identified for ANC-CN did not show any significant pH-dependent shifts, indicating a more stable hydrogen bonding interaction relative to BLC-CN.

Resonance Raman study of cyanide-ligated horseradish peroxidase. Detection of two binding geometries and direct evidence for the "push-pull" effect.

Resonance Raman spectroscopy has been employed to investigate the structure of cyanide adducts of the basic isoenzymes of horseradish peroxidase (HRP) in the pH range 5.5-12.5. Evidence for the binding of cyanide in two forms, characterized by the reversal of ordering of the Fe-CN stretching and Fe-C-N bending vibrations, is observed. Moreover, it is shown that both conformers exhibit an acid-alkaline transition in the pH range employed. In the first conformer, the Fe-C-N linkage is essentially linear, exhibiting axial Fe-CN stretching and Fe-C-N bending frequencies at 453 and 405 cm-1, respectively (at pH 5.5) (Lopez-Garriga et al., 1990). In the second conformer, the Fe-C-N fragment is bent, and the axial stretching and bending modes have been identified at 360 and 422 cm-1 at pH 5.5. At pH 12.5, the v[Fe-CN] stretching mode of the linear conformer shifts down by 9 cm-1 to 444 cm-1 while the bending frequency remains unchanged. For the bent conformer at this pH, the stretching mode shifts to 355 cm-1 (-5 cm-1), and the bending vibration shifts slightly to lower frequency by 2 cm-1 to 420 cm-1. The observed pH-dependent shift of the v[Fe-CN] stretching mode of the linear conformer is attributed to the direct effect of deprotonation of a distal-side amino acid residue while the shift of v[Fe-CN] of the bent conformer is most reasonably ascribable to indirect alteration of the iron-proximal histidine linkage induced by the distal-side deprotonation, a spectral response which reflects a protein-coupled "push-pull" mechanism for heterolytic O-O bond cleavage.