STRUCTURAL INFLUENCE OF CALCIUM ON THE HEME CAVITY OF CATIONIC PEANUTPEROXIDASE AS DETERMINED BY H-1-NMR SPECTROSCOPY

The cationic isozyme of peanut peroxidase (CPRx) is one of many peroxi dases which requires calcium for enzyme activity. It has been previous ly shown that it requires 2 mol calcium to coordinate to 1 mol CPRx, a nd its related peroxidases from the basidiomycete Phanerochaete chryso sporium (LiP) and isozyme C of horseradish (HRPc). X-ray crystallograp hic studies of LiP have shown that calcium is ligated near the C-termi nus of helices proximal and distal to the heme, where it has been sugg ested to maintain the active site. To determine if such a mechanism wa s possible in CPRx, high resolution H-1-NMR spectroscopy was used to s tudy the effect of calcium on the environment of its heme group and th e coordinating histidine residues. The low-spin cyano complex of the e nzyme (CPRxCN) was studied in order to assign the majority of the reso nances arising from the protons in the heme pocket in both the presenc e and absence of bound calcium ions using two dimensional nuclear Over hauser effect spectroscopy (NOESY). The two calcium ions present in CP RxCN were removed by a non-denaturing method and a calcium titration w as performed and monitored by H-1-NMR spectroscopy. These studies show ed that the binding of both calcium ions in CPRx influenced the heme e nvironment in a similar manner (K-d = 0.1 mu M). In particular, calciu m-dependent changes in several heme resonances and the proximal and di stal histidine residues suggest that calcium binding to CPRx causes so me reorientation of these residues with respect to the active site.