There are highly conserved hydrogen bonds between the distal His and the adjacent Asn in many peroxidases. Although the crystal structure of horseradish peroxidase C (HRP) is not available, comparison of the amino acid sequence with cytochrome c peroxidase indicates that Asn70 is making the hydrogen bond with the distal His in the active site of HRP. To investigate the catalytic roles of the hydrogen bond, Asn70 in HRP was replaced with Val (N70V) or Asp (N70D). Though UV-vis, CD, and 1H-NMR spectra of native (plant enzyme), wild-type (recombinant enzyme), and mutant HRPs suggest that the active site and secondary structure are very similar even after mutation, the mutants exhibit low Vmax values for the hydroquinone oxidation (native, 281; wild-type, 283; and N70V, 18; and N70D, 33 microM.min-1). The rates of compound I formation were decreased to less than 10% of that of the native enzyme. The reduction rates of compounds I and II by guaiacol also were reduced to less than 10% of that of the native enzyme. Substituent effects of various phenol derivatives on the reduction of native, wild-type, and mutant compound I were examined. Large negative Hammett rho values (rho N70V:fast = -4.0, rho N70V:slow = -3.6, rho N70D = -3.8, rho native = -6.9, and rho wild-type = -6.8) are an indication of electron transfer being the rate-determining step in the phenol oxidation. However, these results also indicate the participation of the deprotonation step in the compound I reduction process. The proton abstraction from phenol must be harder for the mutants due to the decrease of basicity of the distal His upon mutation. Contrary to phenol oxidation, ABTS [2,2'-azinobis(3-ethybenzothiazoline-6-sulfonic acid)] oxidation activity was substantially increased by the mutations (native, 73; wild-type, 71; N70V, 217; and N70D, 234 microM.s-1). The redox potentials of N70V and N70D compounds II are 957 and 970 mV (vs NHE), which are 95 and 108 mV higher than that of native compound II (862 mV), respectively. Therefore, the high ABTS oxidation activities of mutants are attributed to these high redox potentials of compound II.