Biochemical evidence for heme linkage through esters with Asp-93 and Glu-241 in human eosinophil peroxidase - The ester with Asp-93 is only partiallyformed in vivo

The covalent heme attachment has been extensively studied by spectroscopic methods in myeloperoxidase and lactoperoxidase (LPO) but not in eosinophil peroxidase (EPO). We show that heme linkage to the heavy chain is invariabl y present, whereas heme linkage to the light chain of EPO is present in les s than one-third of EPO molecules. Mass analysis of isolated heme bispeptid es supports the hypothesis of a heme b linked through two esters to the pol ypeptide, Mass analysis of heme monopeptides reveals that >90% have a nonde rivatized methyl group at the position of the light chain linkage. Apparent ly, an ester had not been formed during biosynthesis, The light chain linka ge could be formed by incubation with hydrogen peroxide, in accordance with a recent hypothesis of autocatalytic heme attachment based on studies with LPO (DePillis, G. D., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de M ontellano P. R. (1997) J. Biol. Chem. 272, 8857-8860). By sequence analysis of isolated heme peptides after aminolysis, we unambiguously identified th e acidic residues, Asp-93 of the light chain and Glu-241 of the heavy chain , that form esters with the heme group. This is the first biochemical suppo rt for ester linkage to two specific residues in eosinophil peroxidase. Fro m a parallel study with LPO, we show that Asp-125 and Glu-275 are engaged i n ester linkage. The species with a nonderivatized methyl group was not fou nd among LPO peptides.