Unusual activation during peroxidase reaction of a cytochrome c variant

Cytochrome c has been extensively used as model of peroxidase reaction. The peroxidase activity and stability of a triple mutant CYC-3 (N52I, Y67F and M80A) were studied and compared to those of wild type protein (Wt-16). The CYC-3variant showed ten-fold increased activity in styrene oxidation. An intermediary specie that resembles to Cpd 0 (FeIIIOOH) of peroxidases was detected through EPR measurements during the reaction of CYC-3 with H2O2. Using molecular dynamics (MD) it was found that mutations in CYC-3 induce conformational changes in the M80 loop promoting the rotation of D ring propionate toward heme iron and the inclusion of transient water molecules that could explain the formation of Cpd 0 intermediate. The effects of these conformational changes on the activity increase are discussed.