Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by ¹H‐NMR Spectroscopy

Abstract

The cationic isozyme of peanut peroxidase (CPRx) is one of many peroxidases which requires calcium for enzyme activity. It has been previously shown that it requires 2 mol calcium to coordinate to 1 mol CPRx, and its related peroxidases from the basidiomycete Phanerochaete chrysosporium (Lip) and isozyme C of horseradish (HRPc). X-ray crystallographic studies of Lip have shown that calcium is ligated near the C-terminus of helices proximal and distal to the heme, where it has been suggested to maintain the active site. To determine if such a mechanism was possible in CPRx, high resolution 'H-NMR spectroscopy was used to study the effect of calcium on the environment of its heme group and the coordinating histidine residues. The low-spin cyano complex of the enzyme (CPRxCN) was studied in order to assign the majority of the resonances arising from the protons in the heme pocket in both the presence and absence of bound calcium ions using two dimensional nuclear Overhauser effect spectroscopy (NOESY). The two calcium ions present in CPRxCN were removed by a non-denaturing method and a calcium titration was performed and monitored by 'H-NMR spectroscopy. These studies showed that the binding of both calcium ions in CPRx influenced the heme environment in a similar manner (Kd = 0.1 pM). In particular, calcium-dependent changes in several heme resonances and the proximal and distal histidine residues suggest that calcium binding to CPRx causes some reorientation of these residues with respect to the active site.

The Tightly Bound Calcium of MauG Is Required for Tryptophan Tryptophylquinone Cofactor Biosynthesis

“…Horseradish peroxidase binds two Ca 2þ which are required to maintain the structural integrity of the heme, for which the geometry for catalysis is lost upon Ca 2þ removal (5,6). In cationic peanut peroxidase Ca 2þ is believed to position a catalytically important amino acid around the heme group which also affects the orientation of the porphyrin ring (7).…”

“…Horseradish peroxidase binds two Ca 2+ which are required to maintain the structural integrity of the heme, for which the geometry for catalysis is lost upon Ca 2+ removal , . In cationic peanut peroxidase Ca 2+ is believed to position a catalytically important amino acid around the heme group which also affects the orientation of the porphyrin ring . For manganese peroxidase and lignin peroxidase it has been demonstrated that thermal inactivation which causes distinct alterations in the heme environment, change in heme spin state and changes in overall protein structure, is prevented and reversed in the presence of Ca 2+ , .…”

“…Ca 2þ is required for the activity of enzymes such as phospholipase A 2 (3). Ca 2þ has also been detected in hemecontaining peroxidases of plants, fungi, bacteria, and animals, including canine myeloperoxidase (4), horseradish peroxidase (5,6), cationic peanut peroxidase (7), manganese peroxidase (8), lignin peroxidase (9), and bacterial diheme cytochrome c peroxidase (BCCP) 1 (10). In general, Ca 2þ has been proposed to maintain a heme conformation and heme pocket structure associated with high catalytic activities for these peroxidases.…”

“…Ca 2+ is required for the activity of enzymes such as phospholipase A 2 . Ca 2+ has also been detected in heme-containing peroxidases of plants, fungi, bacteria, and animals, including canine myeloperoxidase , horseradish peroxidase , , cationic peanut peroxidase , manganese peroxidase , lignin peroxidase , and bacterial diheme cytochrome c peroxidase (BCCP) . In general, Ca 2+ has been proposed to maintain a heme conformation and heme pocket structure associated with high catalytic activities for these peroxidases. 1 Abbreviations: MADH, methylamine dehydrogenase; TTQ, tryptophan tryptophylquinone; preMADH, the biosynthetic precursor protein of MADH with incompletely synthesized TTQ; BCCP, bacterial diheme cytochrome c peroxidase; ET, electron transfer; bis-Fe(IV) MauG, redox state of MauG with one heme as Fe(IV)O and the other as Fe(IV); CD, circular dichroism. …”

The modulation of structural stability of horseradish peroxidase as a consequence of macromolecular crowding

“…These Ca 2 + ions do not participate directly in the catalyzed reactions but are key player in supporting native protein structure, consequently guaranteeing the faultless activity and serviceability of enzymes. 5 The basic structural and functional connexions can be unleashed by exploiting the distinct properties of this enzyme making it a prototypical protein for conformational studies. It provides a proficient route for elimination of intracellular hydrogen peroxide from the region by catalyzing oxidation of an array of substrates by organic or hydrogen peroxides.…”