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.

FIGURE 1

The seven-coordinate calcium binding sites in HRPC. The proximal site (purple) is partially solvent-exposed (dotted surface) in a more flexible region of the protein while the distal site (blue) is buried in the protein core. Image from the energy-minimized structure generated using the 1ATJ.PDB coordinates (Gajhede et al., 1997). Solvent-accessible surface (115.92 Å) was calculated using the Connolly algorithm (Connolly, 1983) with a 1.4-Å probe radius.

FIGURE 2

Orientation of the heme for the NSD calculations.

FIGURE 3

Polarized Raman spectra of native Ca²⁺-depleted HRPC (pH 8) excited at 476.5 and 457.9 nm (a) and at 514.5 and 496.5 nm (b), which cover both B-preresonance and Q_v-resonance excitation.

FIGURE 4

Comparison of DPRs for native and Ca²⁺-depleted HRPC: ν₄ (top), ν₂₁ (middle), and ν₁₁ (bottom).

FIGURE 5

Stereo view of the backbone traces of the HRPC (top) and Ca²⁺-depleted (bottom) average structure after 200 ps of molecular dynamics.

FIGURE 6

The effect of calcium removal in the vicinity of the heme of HRPC (left) and Ca²⁺-free HRPC (right). The aromatic cluster consisting of Phe41, 143, 152, and 179 on the distal side and Phe172, 221, 229, and 277 on the proximal side are rendered purple. Pro141 and His42 are yellow, and wat90 is a red sphere.

FIGURE 7

View of the his170 imidazole orientation with respect to the N_pyrr-Fe-N_pyrr line in the hemes extracted from the HRPC models. Clockwise from top left: HRPC + 2Ca²⁺; Ca²⁺-depleted HRPC; HRPC + distal Ca²⁺; and HRPC + distal Ca²⁺.

FIGURE 8

Out-of-plane displacements in Å of the minimal basis set for the hemes in the average structures extracted from the MDS trajectories. Deformations along the six lowest frequency normal coordinates of the following D_4h symmetry types: B_2u (saddling), B_1u (ruffling), A_2u (doming), E_g(x) (x-waving), E_g(y) (y-waving), and A_1u (propellering).