Fifteen years of Raman spectroscopy of engineered heme containing peroxidases: what have we learned?

Spectroscopic techniques have been fundamental to the comprehension of peroxidase function under physiological conditions. This Account examines the contribution to our understanding of heme peroxidases provided by electronic and resonance Raman spectroscopies in conjunction with site-directed mutagenesis. The results obtained over 15 years with several heme peroxidases and selected mutants have provided important insights into the influence exerted by the protein in the vicinity of the active site via key amino acids on the functionality and stability of the enzymes. Moreover, resonance Raman spectroscopy has revealed that a common feature of heme peroxidases is the presence of an extensive network of H-bonds coupling the distal and proximal sides, which has a profound influence on the heme ligation, affecting both the fifth and the sixth coordination sites.