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The Endogenous Calcium Ions of Horseradish Peroxidase C Are Required to Maintain the Functional Nonplanarity of the Heme

Monique Laberge^*, Qing Huang, Reinhard Schweitzer-Stenner and Judit Fidy^*

^* Institute of Biophysics and Radiation Biology, Semmelweis University, Puskin u. 9, Budapest H-1088, Hungary; and Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico PR00931, USA

Correspondence: Address reprint requests to Monique Laberge, Semmelweis University, Dept. of Biophysics and Radiation Biology, P.O. Box 263, Budapest, H-1444, Hungary. Tel.: 36-1-267-6261; Fax: 36-1-266-6656; E-mail: laberge{at}puskin.sote.hu.

Horseradish peroxidase C (HRPC) binds 2 mol calcium per mol of enzyme with binding sites located distal and proximal to the heme group. The effect of calcium depletion on the conformation of the heme was investigated by combining polarized resonance Raman dispersion spectroscopy with normal coordinate structural decomposition analysis of the hemes extracted from models of Ca²⁺-bound and Ca²⁺-depleted HRPC generated and equilibrated using molecular dynamics simulations. Results show that calcium removal causes reorientation of heme pocket residues. We propose that these rearrangements significantly affect both the in-plane and out-of-plane deformations of the heme. Analysis of the experimental depolarization ratios are clearly consistent with increased B_1g- and B_2g-type distortions in the Ca²⁺-depleted species while the normal coordinate structural decomposition results are indicative of increased planarity for the heme of Ca²⁺-depleted HRPC and of significant changes in the relative contributions of three of the six lowest frequency deformations. Most noteworthy is the decrease of the strong saddling deformation that is typical of all peroxidases, and an increase in ruffling. Our results confirm previous work proposing that calcium is required to maintain the structural integrity of the heme in that we show that the preferred geometry for catalysis is lost upon calcium depletion.

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