Infrared Spectra of Carbonyl Horseradish Peroxidase and Its Substrate Complexes: Characterization of pH-Dependent Conformers

Abstract

The FTIR spectra of carbonyl horseradish peroxidase (HRP) were re-investigated over an extended pH range between pH 3 and 11.5. Two ν(CO) bands were observed at 1934 and 1905 cm-1 and the relative absorbance intensity of these bands varied with pH. The absorbance of the 1905-cm-1 band increased in intensity on ionization of a group with a pKa1 = 4.0 ± 0.1, and decreased in intensity on ionization of a second group with a pKa2 = 8.7 ± 0.1. Since the vibrational spectrum of HRP−CO was not recorded below pH 5 previously, pKa1 was not observed and pKa2 was assigned to deprotonation of the distal His42 (Barlow, C. H.; Ohlsson, P. I.; Paul, K. G. Biochemistry 1976, 15, 2225). pKa2 is re-assigned here to a residue involved in the H-bonding network between the distal and proximal heme cavities, and pKa1 to deprotonation of the distal His42, since a pKa <4 has been assigned to this residue in ferric HRP. Parallel pH-dependent changes were observed in the amide I‘ region of HRP−CO, suggesting that shifts in the population of the CO conformers are accompanied by conformational changes in the helices surrounding the heme. Formation of substrate−HRP−CO ternary complexes with substrates of the type Ph−CO−NH−X (X = H, OH, NH2, CH3) resulted in shifting of the FeCO conformation equilibrium to a single form at pH 7.0 with ν(CO) values (cm-1) of 1904 (X = H), 1911 (X = OH), 1916 (X = NH2), and 1900 (X = CH3). Examination of the pH dependence of the FTIR spectrum of the benzhydroxamic acid (BHA; X = OH) ternary complex revealed that the single ν(CO) band at 1911 cm-1 persists between pH 3 and 11, indicating that BHA binding inhibits the pH-dependent conformation equilibria of the FeCO unit. The combined FTIR results on the binary and ternary complexes are consistent with the involvement of Arg38, and not His42, in H-bonding to the CO ligand in HRP.