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 con trol the reaction mechanism and ligand binding has been investigated f urther by analysis of the electronic absorption and resonance Raman (R R) spectra of distal site mutants of recombinant horseradish peroxidas e (HRP-C). The roles of the conserved distal histidine and arginine r esidues, particularly in the context of the catalytic mechanism origin ally proposed for cytochrome c peroxidase (CCP), have been evaluated b y 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 a t neutral pH. In addition, the ferric forms have been studied at alkal ine pH. The relative populations of the three heme spin states charact eristic of HRP-C and its mutants were found to vary markedly from mut ant to mutant, This diversity of heme spin state populations among the various mutants has allowed a well-defined set of RR frequencies to b e compiled for the three heme spin stares. These frequencies support t he analysis of wild-type HRP-C in terms of two heme states, five-(5cH S#) and six-coordinate high-spin (6cHS(#)), which exhibit anomalous RR frequencies compared to those of model heme systems. The third heme s pin state is identified as being six-coordinate high-spin, displaying typical RR frequencies (6cHS). The 6cHS(#) and the 6cHS heme states ar e 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 (6cHS(#)), which exhibit anomalous RR freq uencies compared to those of model heme systems. The third heme spin s tate is identified as being six-coordinate high-spin, displaying typic al RR frequencies (6cHS). The 6cHS(#) and the 6cHS heme states are cha racterized by H bonding between the iron-bound water molecule and the Arg38 residue or the His42 residue, respectively. The proportion of si x-coordinate high-spin heme states is at a minimum in the Arg38Leu mut ant, indicating that the occupancy of the distal water molecule site i s reduced in this mutant. The His42Arg mutant is distinguished from th e other mutants by the unexpected presence of an iron-bound hydroxyl g roup at neutral pH. The spectral changes induced upon complexation wit h BHA indicate that both the distal histidine and arginine are involve d in BHA binding; however, the arginine residue appears to play a more critical role, Measurements at pH 12 suggest there is a concerted inv olvement 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 b etween the roles of these residues in the reaction of H2O2 With the en zyme and the alkaline transition is noted. By comparison with the resu lts from corresponding mutants of CCP, it appears that although the hy drogen-bonding network linking the distal and proximal sides of the he me is conserved the distal cavity in HRP-C differs significantly from that of CCP. However, some similarities in the local environment of th e distal arginine are suggested.