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

Abstract

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 Ca(2+)-bound and Ca(2+)-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 Ca(2+)-depleted species while the normal coordinate structural decomposition results are indicative of increased planarity for the heme of Ca(2+)-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.

The role of Arginine 38 in horseradish peroxidase enzyme revisited: A computational investigation

“…In the second simulated system, R38L-H 2 O 2 , we used exactly the same starting coordinates of the first simulation with Arginine in position 38 substituted with Leucine. Structural Ca 2+ ions were also included in the system because of their documented importance in maintaining the structural integrity of heme (21). The solutes (HRP-H 2 O 2 and R38L-H 2 O 2 ) were put at the centre of a rectangular box (74.5x87.2x69.7 Å 3 ) filled with Single Point Charge (22) (SPC) water molecules at a density of 1000 kg/m 3 (13371 SPC water molecules).…”

Calcium Salts Enhance Activity and Azo Dye Decolourisation Capacity of Crude Peroxidase from <i>Armoracia rusticana</i>

“…It has been demonstrated that in the presence of calcium, the free-energy during unfolding of native HRP C is much greater than in the absence of calcium. This shows that calcium enhances the stability of the enzyme [23]. The presence of calcium is also essential for the proper folding of HRP C [24].…”

Computational studies on a new cationic peroxidase isoenzyme from artichoke leaves

“…16 In addition, a structure prediction was performed also for CysPrx2. The corresponding model was generated by I-TASSER with the restraint that the eight cysteines forming the disulphide bridges (which are conserved in CysPrx2) are in contact.…”