Abstract
The identity of the non-extractable heme of mammalian lactoperoxidase (LPO) has remained unsolved for over 40 years. Accepted possibilities include a constrained heme b or an 8-thiomethylene-modified heme b. Recent studies of myeloperoxidase (MPO) (Fenna, R., Zeng, J., and Davey, C. (1995) Arch. Biochem. Biophys. 316, 653-656; Taylor, K. L., Strobel, F., Yue, K. T., Ram, P., Pohl, J., Woods, A. S., and Kinkade, J. M., Jr. (1995) Arch. Biochem. Biophys. 316, 635-642) suggest possible prosthetic group similarities between MPO and LPO. To address heme identity for LPO, we used comparative magnetic circular dichroism (MCD) spectroscopy of LPO versus myoglobin (Mb), horseradish peroxidase (HRP), and MPO. MCD spectra of native Fe3+-LPO and Fe3+-CN--LPO are approximately 10 nm red shifted from analogous forms of Mb and HRP, including the formate-Mb adduct. MCD spectra of native LPO and MPO are opposite in sign, and MCD spectra of their cyanoadducts also differ. These data indicate the LPO heme is distinct from heme b of Mb and HRP as well as from "heme m" of MPO. From this work and literature analysis, we suggest that the non-extractable "heme l" of LPO has the two vinyl groups of heme b but lacks the 2-sulfonium-vinyl linkage of heme m. The observed red shifts in LPO spectra may derive from ester linkages to protein as for MPO. Strong spectral analogies between LPO and mammalian peroxidases (e.g. from saliva, eosinophils, thyroid, intestine) indicate similar prosthetic heme moieties.
Highlights
Lactoperoxidase (LPO,1 EC 1.11.1.7) is a mammalian peroxidase that is the product of exocrine gland secretion, e.g. into milk, saliva, and tears (1–3)
magnetic circular dichroism (MCD) spectra of native Fe3؉-LPO and Fe3؉CN؊-LPO are ϳ10 nm red shifted from analogous forms of Mb and horseradish peroxidase (HRP), including the formate-Mb adduct
Given the high degree of spectral and protein sequence agreement between mammalian peroxidases (8, 9, 19), we suggest that the heme l of LPO, or a very closely related heme, is likely to be the heme of thyroid peroxidase (TPO), salivary peroxidase (SPO), eosinophil peroxidase, and other mammalian peroxidases
Summary
(Received for publication, July 17, 1995, and in revised form, November 20, 1995). Laura A. Fenna and colleagues (9) and Taylor et al (19) have determined the structure of the “heme m” prosthetic group of native MPO (Fig. 1C) These studies clearly identified the prosthetic group as an iron porphyrin, resolving a long standing controversy where unusual spectral properties of MPO were suggested to derive from an (atypical) iron chlorin prosthetic moiety (for example, Ref. 13 and references therein). Our approach is comparative spectral analysis of LPO and the analogous forms of: 1) Mb and HRP, both of which have heme b and, respectively, either N–His or N–HisϪ 5th ligands; and 2) MPO, which has the novel heme m and N–HisϪ ligation (9, 19) These proteins were examined in both their native and in the Fe3ϩCNϪ forms, using MCD and electronic absorption spectroscopy. Given the high degree of spectral and protein sequence agreement between mammalian peroxidases (8, 9, 19), we suggest that the heme l of LPO, or a very closely related heme, is likely to be the heme of TPO, SPO, eosinophil peroxidase, and other mammalian peroxidases
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