Effects of a highly conserved hydrogen bond between the distal histidine (His) and a nearby asparagine residue (Asn) of peroxidases upon the active site structures were investigated using resonance Raman (RR) and EPR spectroscopy. Although there is no crystallographic data for horseradish peroxidase (HRP), Asn70 is deduced to be the hydrogen bond acceptor for HRP. Accordingly, site-directed mutagenesis of Asn70 to Val (N70V) and to Asp (N70D) was carried out with HRP, and their RR spectra were compared with those of native and wild type (WT) enzymes in the resting, reduced, CN-bound ferric and compound II states. In the resting state, the six-coordinate high-spin structure is the main component for N70V and N70D mutants, while the five-coordinate high-spin structure is dominant for the native and WT HRPs. This was confirmed with EPR spectra. The Fe-III-CN stretching (nu(Fe-CN)) and bending RR bands of the linear and bent forms were identified using (CN)-C-12-N-15 and (CN)-C-13-N-14 isotopes. The nu(Fe-CN) frequency of the linear form is lower for the mutants than for native enzyme, and the spectral patterns of the mutants at pH 7.0 resemble that of the basic form of native HRP. The Fe-histidine stretching bands of reduced HRPs exhibit pH dependent frequency shifts, and the midpoint pH values were 7.2, 5.9, and 5.5 for native, N70V, and N70D, respectively. This change is ascribed to the acid-base transition of the distal His. While the Fe-IV=0 stretching (nu(Fe=0)) frequency of compound LI at pD 7.0 is lower than that at pD 10.0 for native enzyme, the nu(Fe=0) band of the mutants show no pH dependent frequency shifts between pD 7.0 and pD 10.0. However, the H2O/D2O frequency change of nu(Fe=0) and the oxygen atom exchange with bulk water suggested the presence of the hydrogen bond between the oxygen ligand of the ferryloxo heme and distal His for these mutants at pD 10.0. On the basis of these observations, it is proposed that the hydrogen bond between the N-delta-proton of distal His and Asn70 regulates the pK(a) of the N-epsilon- proton (and thus the reactivity of compound LI at the distal side) and also affects the Fe-His bond at the proximal side via tertiary structure changes.