INFRARED SPECTROSCOPIC STUDIES OF CARBONYL HORSERADISH PEROXIDASES

IR difference spectra, FeIICO vs. FeIII, of horseradish peroxidase isoenzymes A2 and C were recorded from 2000-1800 cm-1. Under alkaline conditions, pH 9, both isoenzymes exhibited 2 CO stretching bands, at 1938 and 1925 cm-1 for A2 and at 1933 and 1929 cm-1 for C. As the pH was lowered, the low-frequency band for each isoenzyme decreased in intensity with a concommitant appearance and increase in intensity of a band of 1906 and 1905 cm-1 for the A2 and C isoenzymes, respectively. These changes conformed to pK values of 6.7 for the A2 and 8.8 for the C isoenzymes of horseradish peroxidase. The interpretation of the IR results was simplified by the observation that a linear relationship existed between the redox potential, Em7, for the FeIII/FeII system vs. the IR CO stretching frequency, .nu.CO, for cytochrome a3, Hb, myoglobin and cytochrome P-450cam with substrate. The primary force altering .nu.CO in these heme proteins is apparently a variation in electron density at the heme Fe and not direct protein interactions with the CO ligand. The horseradish peroxidase IR bands in the 1930-cm-1 region correlated well with this relationship. The large deviation of the 1905-cm-1 band from the linear relationship and its dependence upon hydrogen ion concentration were consistent with horseradish peroxidase having a single CO binding site which can hold in 2 geometries, one of which contained an amino acid moiety capable of forming a H bond to the carbonyl oxygen.