Fourier Transform Infrared Spectroscopic Investigation of the Binary and Ternary Cyanide Adducts of the Oxidized Horseradish Peroxidase: Identification of a Second Stretching Mode for the Carbon-Nitrogen Bond of the Bound Cyanide Ion

Abstract

ABSTRACT The binary and ternary cyanide adducts of the ferric horseradish peroxidase were investigated by Fourier transform infrared spectroscopy. The carbon-nitrogen bond of the bound cyanide ion in the binary ferric cyanohorseradish peroxidase exhibits two stretching vibrations at 2130 cm−1 and 2127 cm−1 with the latter mode being observed in this work for the first time. This finding supports the results of the resonance Raman study of cyanohorseradish peroxidase, which identified two iron-carbon-nitrogen bending vibrations and two iron-carbon stretching vibrations, proving the existence of two conformational states. The identification of the latter carbon-nitrogen stretching frequency allowed the assignment of all of the vibrational modes of the iron-carbon-nitrogen groups of the two conformational states of the ferric cyanohorseradish peroxidase. The first conformer is characterized by a carbon-nitrogen stretch at 2130 cm−1, an iron-carbon stretch at 453 cm−1, and an iron-carbon-nitrogen bending mode at 405 cm−1. The second state has a carbon-nitrogen stretch at 2127 cm−1, an iron-carbon stretch at 360 cm−1, and an iron-carbon-nitrogen bending mode at 422 cm−1. The iron-carbon stretching band is weakly sensitive to pH changes, but it is sensitive to H2O/D2O substitution, indicating that the bound cyanide ion in cyanohorseradish peroxidase is hydrogen bonded to the surrounding protein. The two states were attributed to variation in the extent of hydrogen bonding of the iron-carbon-nitrogen groups in the two states. The carbon-nitrogen stretching vibrations of the ternary complexes of cyanohorseradish peroxidase with ferulic acid, benzamide, and benzhydroxamic acid have been investigated for the first time. The binding of the substrate to cyanohorseradish peroxidase does not always lead to the vanishing of one of the conformational states as in the carbon monoxide adducts of the ferrous horseradish peroxidase, but can cause shifts in the νC-N frequency and in the relative population of both conformational states.