The extent to which the structural Ca2+ 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 Ca2+ ion. The Fe(III) heme states are modified upon Ca2+ depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca2+-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 Ca2+ depletion are easily reversed. Contrary to the native protein, the Ca2+-depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the v(Fe-Im) stretching mode downshifts 27 cm(-1) upon Ca2+ depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca2+-depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca2+ depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca2+ ion as that which is lost upon Ca2+ depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.

The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase / B.D.Howes; A. Feis; L. Raimondi; C.Indiani; G. Smulevich. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - STAMPA. - 276:(2001), pp. 40704-40711. [10.1074/jbc.M107489200]

The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase

HOWES, BARRY DENNIS;FEIS, ALESSANDRO;RAIMONDI, LAURA;SMULEVICH, GIULIETTA
2001

Abstract

The extent to which the structural Ca2+ 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 Ca2+ ion. The Fe(III) heme states are modified upon Ca2+ depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca2+-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 Ca2+ depletion are easily reversed. Contrary to the native protein, the Ca2+-depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the v(Fe-Im) stretching mode downshifts 27 cm(-1) upon Ca2+ depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca2+-depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca2+ depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca2+ ion as that which is lost upon Ca2+ depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.
2001
276
40704
40711
B.D.Howes; A. Feis; L. Raimondi; C.Indiani; G. Smulevich
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/309741
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