COMPARATIVE H-1-NMR STUDY OF FERRIC LOW-SPIN CYTOCHROME-C PEROXIDASE AND HORSERADISH-PEROXIDASE

Proton NMR spectra of the cyanide complexes of cytochrome c peroxidase from baker's yeast (CcP) and recombinant protein expressed in Escherichia coli (MKT-IGCcP) have been recorded in water. The paramagnetically shifted exchangeable proton signals have been assigned through NOE and NOESY experiments. NOESY and COSY experiments in D2O and H2O buffers have allowed us to extend the assignments to heme substituents and other residues. Comparison is made with the analogous spectrum of the cyanide adduct of horseradish peroxidase (HRP) (Thanabal, V.; de Ropp, J. S.; La Mar, G. N. J. Am. Chem. Soc. 1988, 110, 3027). HRP-CN- and CcP-CN- systems are highly analogous as shown by the similarity of the pseudocontact shifts of the distal histidine protons and by the protonation status of the proximal and distal histidine in both systems. A more extensive exchangeable proton connectivity network is observed in CcP-CN-. Small but significant structural differences have been identified in the orientation of the proximal histidyl residues of HRP-CN- and CcP-CN- and in the interresidue distances in the distal heme pocket of CcP-CN- and MKT-IGCcP-CN- as a function of the identity of the residue present at position 53.