Abstract
The extent to which the structural Ca(2+) ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca(2+) ion. The Fe(III) heme states are modified upon Ca(2+) depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca(2+)-depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca(2+) depletion are easily reversed. Contrary to the native protein, the Ca(2+)-depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the nu(Fe-Im) stretching mode downshifts 27 cm(-1) upon Ca(2+) depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca(2+)-depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca(2+) depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca(2+) ion as that which is lost upon Ca(2+) depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.
Highlights
Peroxidases of the plant peroxidase superfamily are hemecontaining enzymes that oxidize a variety of aromatic molecules in the presence of hydrogen peroxide
Reconstitution leads to a rise in the specific activity, achieving 85% that of native horseradish peroxidase isoenzyme C (HRPC)
Graph on gel filtration (Fig. 2A). This implies that 1 mol of calcium per mol of enzyme is present in the Ca2ϩ-depleted protein instead of the 2 mol of calcium per mole of enzyme present in native HRPC (4)
Summary
2.1 Ϯ 0.1 6.0 Ϯ 0.6 a The percentages quoted for the activity (measured with ABTS) are with respect to native protein. In the preparation of both Ca2ϩ-depleted and reconstituted HRPC, identical results were obtained using a 23 ϫ 1-cm column of Sephacryl S100 high resolution gel (Amersham Pharmacia Biotech). The dissociation constant, Kd, for BHA binding to Ca2ϩ-depleted HRPC in a 5 mM EDTA, 50 mM Tris/HCl solution at pH 7.8, 25 °C was determined following the procedure of Smith and co-workers (15, 16). Enzyme concentrations were determined spectrophotometrically using extinction coefficients of 112 and 107 mMϪ1 cmϪ1 at the Soret maxima for native and Ca2ϩ-depleted HRPC, respectively (Table I). For each enzyme (native, Ca2ϩ-depleted, and reconstituted HRPC), the value of Vmax was determined for three different concentrations, and the values were plotted to determine the resultant slope by regression analysis. Peak intensities were determined using a curve-fitting program to simulate experimental spectra with Lorentzian line shapes
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