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Keywords:

  • resonance Raman spectroscopy;
  • peroxidase;
  • heme distortion;
  • site-directed mutants;
  • symmetry-lowering

Abstract

Resonance Raman (RR) spectra have been acquired for human myeloperoxidase (MPO), and its Met243Thr and Asp94Val mutants with different excitation wavelengths and in polarized light. The proteins were characterized as ferric, ferrous and ferric–CN complexes in order to study the heme configuration in various coordination, spin and oxidation states. Well-defined spectra of the five-coordinate high spin (ferrous), six-coordinate high spin (ferric) and low spin (ferric–CN) species were obtained. The data allowed us to propose an almost complete assignment of the RR bands. The richness of the RR spectra of MPO is because of the activation of almost all the in-plane skeletal modes observed for the Ni–octaethylporphyrin model compound, induced by the distortion of the heme imposed by the covalent links with the protein. The two mutants, which lost at least one of the covalent links between the protein and the heme group, were useful to determine the effect of the symmetry lowering of the heme group. Copyright © 2006 John Wiley & Sons, Ltd.