Mutation of distal residues of horseradish peroxidase: influence on substrate binding and cavity properties.

The manner in which the distal heme pocket residues of peroxidases control the reaction mechanism and ligand binding has been investigated further by analysis of the electronic absorption and resonance Raman (RR) spectra of distal site mutants of recombinant horseradish peroxidase (HRP-C*). The roles of the conserved distal histidine and arginine residues, particularly in the context of the catalytic mechanism originally proposed for cytochrome c peroxidase (CCP), have been evaluated by studying the His42 --> Leu, His42 --> Arg, Arg38 --> Gly, and Arg38 --> Leu variants of HRP-C*. Spectra of the ferric forms, their complexes with benzohydroxamic acid (BHA), and the ferrous forms have been recorded at neutral pH. In addition, the ferric forms have been studied at alkaline pH. The relative populations of the three heme spin states characteristic of HRP-C* and its mutants were found to vary markedly from mutant to mutant. This diversity of heme spin state populations among the various mutants has allowed a well-defined set of RR frequencies to be compiled for the three heme spin states. These frequencies support the analysis of wild-type HRP-C* in terms of two heme states, five- (5cHS#) and six-coordinate high-spin (6cHS#), which exhibit anomalous RR frequencies compared to those of model heme systems. The third heme spin state is identified as being six-coordinate high-spin, displaying typical RR frequencies (6cHS). The 6cHS# and the 6cHS heme states are characterized by H bonding between the iron-bound water molecule and the Arg38 residue or the His42 residue, respectively. The proportion of six-coordinate high-spin heme states is at a minimum in the Arg38Leu mutant, indicating that the occupancy of the distal water molecule site is reduced in this mutant. The His42Arg mutant is distinguished from the other mutants by the unexpected presence of an iron-bound hydroxyl group at neutral pH. The spectral changes induced upon complexation with BHA indicate that both the distal histidine and arginine are involved in BHA binding; however, the arginine residue appears to play a more critical role. Measurements at pH 12 suggest there is a concerted involvement of both distal residues in mediating the alkaline transition of HRP-C*. Arg38 appears to be essential for stabilization of the OH- ligand, while His42 acts as a H bond acceptor. A striking similarity between the roles of these residues in the reaction of H2O2 with the enzyme and the alkaline transition is noted. By comparison with the results from corresponding mutants of CCP, it appears that although the hydrogen-bonding network linking the distal and proximal sides of the heme is conserved the distal cavity in HRP-C differs significantly from that of CCP. However, some similarities in the local environment of the distal arginine are suggested.