Fine-tuning of the binding and dissociation of CO by the amino acids of the heme pocket of Coprinus cinereus peroxidase

Resonance Raman and infrared spectra and the CO dissociation rates (k(off)) were measured in Coprinus cinereus peroxidase (CIP) and several mutants in the heme binding pocket. These mutants included the Asp245Asn, Arg51Leu, Arg51Gln, Arg51Asn, Arg51Lys, Phe54Trp, and Phe54Val mutants. Binding of CO to CIP produced different CO adducts at pH 6 and 10. At pH 6, the bound CO is H-bonded to the protonated distal His55 residue, whereas at alkaline pH, the vibrational signatures and the rate of CO dissociation indicate a distal side which is more open or flexible than in other plant peroxidases. The distal Arg51 residue is important in determining the rate of dissociation in the acid form, increasing by 8-17-fold in the Arg51 mutants compared to that for the wild-type protein. Replacement of the distal Phe with Trp created a new acid form characterized by vibrational frequencies and k(off) values very similar to those of cytochrome c peroxidase.