Binding of Iodide to Arthromyces ramosus Peroxidase Investigated with X-ray Crystallographic Analysis, 1H and 127I NMR Spectroscopy, and Steady-state Kinetics

Keiichi Fukuyama,Koichi Sato,Hiroyuki Itakura,Seizo Takahashi,Toichiro Hosoya

doi:10.1074/jbc.272.9.5752

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.

Highlights

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
The x-ray crystallographic analysis reported here shows that the ARP-iodide complex contains only one iodine atom, which is located at the entrance of the access channel to the distal side of the heme, 12.8 Å away from the heme iron (Fig. 1, Fig. 2, and Table III)
This is the first report on the binding site of the electron donor molecule to peroxidase determined by x-ray crystallographic analysis

Summary

EXPERIMENTAL PROCEDURES

Materials—ARP that had been purified by the method of Morita et al [27] was provided by Dr T. Diffraction data for the iodide derivative were collected to 2.06 Å resolution at room temperature on an R-AXIS IIc imaging plate area detector. One was an (Fo Ϫ Fc) synthesis at 2.06 Å resolution, where Fo is the observed structure factor of the iodide derivative and Fc the calculated structure factor derived from the atomic parameters of the native crystal. Typical spectra were obtained by 200,000 – 800,000 transients using 5-degree pulse at 32,000 data points over a 41.6-kHz bandwidth and applied 100-Hz line broadening to the TABLE I Conditions and results of intensity measurement. Peroxidase Kinetics—The rate of oxidation of iodide ion by H2O2 was measured at 295 K by following I3Ϫ at 350 nm as described previously [39], except that the total volume of the reaction mixture was reduced to 2.4 ml and the H2O2 concentration was 270 ␮M. The spectrophotometer used was a Hitachi model UV-3000 spectrophotometer equipped with a thermoregulator made of Peltier units, and the time course was recorded on a floppy disk to instantly obtain the initial rate

RESULTS

DISCUSSION

Fe of Heme

The next step would be electron transfer from the imidazole