Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the involvement of two key distal heme cavity residues, histidine 42 and arginine 38, in the formation and structure of the carbon monoxide complex of HRPC (carbonyl HRPC). The rates of CO binding to the wild-type glycosylated and non-glycosylated recombinant (HRPC*) ferrous enzymes were essentially identical and exhibited the same pH dependence with pKas at 7.4 and 4.0. Data obtained with the His-42 → Leu [(H42L)HRPC*)] and Arg-38 → Leu [(R38L)HRPC*] mutants allowed the pKa at 7.4 in ferrous HRPC to be assigned to His-42. The infra-red and electronic absorption spectra of HRPC-CO, HRPC*-CO, (R38L)HRPC*-CO and (H42L)HRPC*-CO have been investigated over the pH range 3.0–10.0. HRPC*-CO exhibited two ν (CO) bands at 1934 cm–1 and 1905 cm–1 whose relative intensity changed with pH, showing an acidic and a basic pKa as previously reported for HRPC [IE Holzbaur; AM English, AA Ismail (1996) J Am Chem Soc 118 : 3354–3359]. (H42L)HRPC*-CO and (R38L)HRPC*-CO exhibited single infra-red bands at 1924.2 cm–1 (pH 7.0) and 1941.5 cm–1 (pH 5.0) respectively. Acidic and alkaline pKas were determined from shifts in the infra-red frequencies and by UV-visible spectrophotometry at the Söret maxima. (H42L)HRPC*-CO exhibited a pKa at ∼pH 4.0 but no alkaline pKa. (R38L)HRPC*-CO exhibited a single pKa at pH 6.5. Shifts of 2–3 cm–1 in ν (CO) with (H42L)HRPC*-CO in D2O show that a distal residue is H-bonding to the CO in this variant at both pD 7.5 and 3.9. However, with (R38L)HRPC*-CO, only a small shift of the ν (CO) band was observed at pD 5.5. The results are consistent with the involvement of Arg-38 in H-bonding to the CO ligand in HRPC and with His-42 modulating the distribution of carbonyl HRPC conformers below pH 8.7. These data are discussed in terms of the importance of distal pocket polarity in HRPC. It is concluded that His-42 can have a pKa between 4.0 and 8.7 depending on its environment and the nature of the distal ligand at position 38. This enables His-42 to carry out multiple functions during the catalytic cycle of HRPC.