Identification of the fifth axial heme ligand of chloroperoxidase.
Chloroperoxidase from Caldariomyces fumago catalyzes the peroxidative chlorination of organic acceptor molecules. From a variety of spectroscopic data, it had long been thought that chloroperoxidase possessed an active site structure similar to that of cytochrome P-450cam. Resonance Raman studies conducted with isotopically substituted enzyme proved conclusively that the fifth axial ligand to the ferriprotoporphyrin IX moiety of chloroperoxidase is indeed a cysteine thiolate (Bangcharoenpaurpong, O., Champion, P. M., Hall, K. S., and Hager, L. P. (1986) Biochemistry 25, 2374-2378). In this study, Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was used to ascertain which of the 3 cysteine residues in the primary structure of chloroperoxidase serves as the fifth axial heme ligand; two of the cysteine residues were earlier shown to be involved in a disulfide linkage. Apoprotein was labeled under denaturing conditions with 5,5'-dithiobis(2-nitrobenzoic acid). A unique peptide, containing the thionitrobenzoate adduct, was isolated via reverse phase HPLC following digestion with endoproteinase Glu-C. Amino acid and Edman sequence analysis revealed the fifth axial ligand in chloroperoxidase to be cysteine 29. Under reducing and denaturing conditions, incubation of apochloroperoxidase with Ellman's reagent resulted in 3 labeled residues. Proteolysis and isolation of the labeled peptides using reverse phase HPLC and subsequent Edman sequence analysis detected and identified the thionitrobenzoate adducts of each of the three cysteinyl peptides of chloroperoxidase.
Blanke
S R
SR
Department of Biochemistry, University of Illinois, Urbana 61801.
Hager
L P
LP
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002711:Chloride Peroxidase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000378:metabolism*; D013059:Spectrum Analysis, Raman
D000596:Amino Acids; D006418:Heme; D004228:Dithionitrobenzoic Acid; D010544:Peroxidases; D002711:Chloride Peroxidase