The FTIR spectra of carbonyl horseradish peroxidase (HRP) were re-investigated over an extended pH range between pH 3 and 11.5. Two v(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 pK(a1) = 4.0 +/- 0.1, and decreased in intensity on ionization of a second group with a pK(a2) = 8.7 +/- 0.1. Since the vibrational spectrum of HRP-CO was not recorded below pH 5 previously, pk(a1) was not observed and pK(a2) was assigned to deprotonation of the distal His42 (Barlow, C. H.; Ohlsson, P. I.; Paul, K. G. Biochemistry 1976, 15, 2225). pK(a2) is re-assigned here to a residue involved in the H-bonding network between the distal and proximal heme cavities, and pK(a1) to deprotonation of the distal His42, since a pK(a) < 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 v(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 v(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.