Identification of the fifth axial heme ligand of chloroperoxidase.

S. R. Blanke, L. P. Hager

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)18739-18743
Number of pages5
JournalThe Journal of biological chemistry
Issue number35
StatePublished - Dec 15 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Identification of the fifth axial heme ligand of chloroperoxidase.'. Together they form a unique fingerprint.

Cite this