SOLUTION CHARACTERIZATION BY NMR-SPECTROSCOPY OF 2 HORSERADISH-PEROXIDASE ISOENZYME-C MUTANTS WITH ALANINE REPLACING EITHER PHE142 OR PHE143

Site-directed mutagenesis of the horseradish peroxidase isoenzyme C (HRP C) gene has been undertaken in order to provide two recombinant enzymes where alanine replaces either Phe142 or Phe143 ([F142A]HRP C and [F143A]HRP C, respectively). These heme enzymes have been characterised in solution using proton NMR spectroscopy for both the high-spin resting and low-spin cyanide-ligated states. Comparison of their NMR spectra with those recorded for wild-type plant HRP C indicates that both the protein fold and the structure of the heme pocket are maintained. The structural integrity of the aromatic donor molecule binding site is altered as a result of the substitution of Phe142 by Ala, but not by the corresponding substitution at Phe 143. This is evident from analysis of perturbations to the chemical shift and linewidth parameters of the proton resonances of two Phe side chains, Phe A and Phe B. that participate in this site. The resting and cyanide-ligated states of [F142A]HRP C bind the aromatic donor molecule, benzhydroxamic acid, three to four times more weakly than the analogous states of wild-type plant HRP C. A titration of cyanide-ligated [F142A]HRP C with benzhydroxamic acid, monitored by NMR spectroscopy, further reveals that the dynamics of complex formation are considerably altered, in that only one of the two possible benzhydroxamic acid binding modes established for the cyanide-ligated wild-type enzyme is significantly populated. Although the assignment of Phe A and Phe B cannot be made to either Phe142 or Phe143, the results confirm that Phe142 is an important, although indirect, determinant of aromatic donor molecule binding and dynamics. The role of phenylalanine side chains in the binding of aromatic donor molecules by heme peroxidases is discussed in the light of these observations and a recent structural model fur HRP C.