Two-dimensional NMR study of the heme active site structure of chloroperoxidase.

The heme active site structure of chloroperoxidase (CPO), a glycoprotein that displays versatile catalytic activities isolated from the marine mold Caldariomyces fumago, has been characterized by two-dimensional NMR spectroscopic studies. All hyperfine shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues including the heme iron ligand (Cys(29)) attributable to the unique catalytic properties of CPO have been firmly assigned through (a) measurement of nuclear Overhauser effect connectivities, (b) prediction of the Curie intercepts from both one- and two-dimensional variable temperature studies, (c) comparison with assignments made for cyanide derivatives of several well characterized heme proteins such as cytochrome c peroxidase, horseradish peroxidase, and manganese peroxidase, and (d) examination of the crystal structural parameters of CPO. The location of protein modification that differentiates the signatures of the two isozymes of CPO has been postulated. The function of the distal histidine (His(105)) in modulating the catalytic activities of CPO is proposed based on the unique arrangement of this residue within the heme cavity. Contrary to the crystal state, the high affinity Mn(II) binding site in CPO (in solution) is not accessible to externally added Mn(II). The results presented here provide a reasonable explanation for the discrepancies in the literature between spectroscopists and crystallographers concerning the manganese binding site in this unique protein. Our study indicates that results from NMR investigations of the protein in solution can complement the results revealed by x-ray diffraction studies of the crystal form and thus provide a complete and better understanding of the actual structure of the protein.