Binding of iodide to Arthromyces ramosus peroxidase investigated with X-ray crystallographic analysis, 1H and 127I NMR spectroscopy, and steady-state kinetics.

Abstract The site and characteristics of iodide binding to Arthromyces ramosus peroxidase were examined by x-ray crystallographic analysis, 1H and 127I NMR, and kinetic studies. X-ray analysis of an A. ramosus peroxidase crystal soaked in a KI solution at pH 5.5 showed that a single iodide ion is located at the entrance of the access channel to the distal side of the heme and lies between the two peptide segments, Phe90-Pro91-Ala92 and Ser151-Leu152-Ile153, 12.8 A from the heme iron. The distances between the iodide ion and heme peripheral methyl groups were all more than 10 A. The findings agree with the results obtained with 1H NMR in which the chemical shift and intensity of the methyl groups in the hyperfine shift region of A. ramosus peroxidase were hardly affected by the addition of iodide, unlike the case of horseradish peroxidase. Moreover, 127I NMR and steady-state kinetics showed that the binding of iodide depends on protonation of an amino acid residue with a pKa of about 5.3, which presumably is the distal histidine (His56), 7.8 A away from the iodide ion. The mechanism of electron transfer from the iodide ion to the heme iron is discussed on the basis of these findings.