THE HEME PROSTHETIC GROUP OF LACTOPEROXIDASE - STRUCTURAL CHARACTERISTICS OF HEME-1 AND HEME-1-PEPTIDES

The heme prosthetic group from the bovine milk enzyme lactoperoxidase (LPO), termed heme 1, is isolated through an approach that combines pr oteolytic hydrolysis and reverse-phase high performance liquid chromat ographic separation of the resulting digest. Application of different proteases yields either a peptide-bound heme (with trypsin and chymotr ypsin) or a peptide-free heme (with proteinase K). Both heme 1 and hem e 1-peptide species were investigated by paramagnetic H-1 NMR spectros copy, electrospray mass spectrometry, and peptide sequence analysis. P aramagnetic H-1 MMR experiments on the low spin bis(cyano)-Fe(III)heme 1 complex conclusively define the heme 1 structure as a 1,5-bis(hydro xymethyl) derivative of heme b. The electrospray mass spectrum of heme 1 confirms the two-site hydroxyl functionalization on this heme, Para magnetic H-1 NMR spectra of the high spin bis(dimethyl sulfoxide)-Fe(I II) complexes of the isolated heme species provide information regardi ng peptide content. Sequence analyses of peptides released from two he me 1-peptide species by base hydrolysis suggest that heme-protein este r linkages in lactoperoxidase occur between the two hydroxyl groups of heme 1 and the carboxylic side chains of glutamate 275 and aspartate 125, These results confirm the earlier reported structural proposal (R ae, T, D., and Goff, H. M. (1996) J. Am. Chem. Soc. 118, 2103-2104).