Comparison of the heme structures of horseradish peroxidase compounds X and II by resonance Raman spectroscopy.

Horseradish peroxidase will catalyze the chlorination of certain substrates by sodium chlorite through an intermediate known as compound X. A chlorite-derived chlorine atom is known to be retained by compound X and has been proposed to be located at the heme active site. Although several heme structures have been proposed for compound X, including an Fe(IV)-OCl group, preliminary data previously reported by our laboratory suggested that compound X contained a heme Fe(IV) = O group, based on the similarity of a compound X resonance Raman band at 788 cm-1 to resonance Raman Fe(IV) = O stretching vibrations recently identified for horseradish peroxidase compound II and ferryl myoglobin. Isotopic studies now confirm that the 788 cm-1 resonance Raman band of compound X is, in fact, due to a heme Fe(IV) = O group, with the oxygen atom derived from chlorite. The Fe(IV) = O frequency of compound X, of horseradish peroxidase isoenzymes B and C, undergoes a pH-induced frequency shift, with behavior which appears to be the same as that previously reported for compound II, formed from the same isoenzymes. These observations strongly suggest that compounds II and X have very similar, if not identical, heme structures. The chlorine atom thus appears not to be heme-bound and may rather be located on an amino acid residue. The studies on compound X reported here were done in a pH region above pH 8, where compound X is moderately stable. The present results do not necessarily apply to compound X below pH 8.

Heme-linked ionization of horseradish peroxidase compound II monitored by the resonance Raman Fe(IV)=O stretching vibration.

Fe(IV)=O resonance Raman stretching vibrations were recently identified by this laboratory for horseradish peroxidase compound II and ferryl myoglobin. In the present report it is shown that Fe(IV)=O stretching frequency for horseradish peroxidase compound II will switch between two values depending on pH, with pKa values corresponding to the previously reported compound II heme-linked ionizations of pKa = 6.9 for isoenzyme A-2 and pKa = 8.5 for isoenzyme C. Similar pH-dependent shifts of the Fe(IV)=O frequency of ferryl myoglobin were not detected above pH 6. The Fe(IV)=O stretching frequencies of compound II of the horseradish peroxidase isoenzymes at pH values above the transition points were at a high value approaching the Fe(IV)=O stretching frequency of ferryl myoglobin. Below the transition points the horseradish peroxidase frequencies were found to be 10 cm-1 lower. Frequencies of the Fe(IV)=O stretching vibrations of horseradish peroxidase compound II for one set of isoenzymes were found to be sensitive to deuterium exchange below the transition point but not above. These results were interpreted to be indicative of an alkaline deprotonation of a distal amino acid group, probably histidine, which is hydrogen bonded to the oxyferryl group below the transition point. Deprotonation of this group at pH values above the pKa disrupts hydrogen bonding, raising the Fe(IV)=O stretching frequency, and is proposed to account for the lowering of compound II reactivity at alkaline pH. The high value of the Fe(IV)=O vibration of compound II above the transition point appears to be identical in frequency to what is believed to be the Fe(IV)=O vibration of compound X.

Observation of the FeIV=O stretching vibration of ferryl myoglobin by resonance Raman spectroscopy.

We have directly observed the oxyferryl group of ferryl myoglobin by resonance Raman spectroscopy. The FeIV = O stretching vibration is observed at 797 cm-1 and confirmed by an 18O-induced isotopic shift to 771 cm-1. The porphyrin center-to-nitrogen distance of ferryl myoglobin is significantly less than that previously observed for horseradish peroxidase compound II, which also contains an FeIV = O heme. The FeIII-CN- stretch of myoglobin (FeIII) cyanide is observed at 454 cm-1, which shifts to 449 cm-1 upon substitution with [13C]cyanide.