Isolation and identification of a protoheme IX derivative released during autolytic cleavage of human myeloperoxidase

Myeloperoxidase (MPO) is a functionally important component of the normal human neutrophil host defense system. This enzyme possesses a dimeric structure composed of two heavy-subunit/light-subunit protomers, with a heme-like prosthetic group covalently linked to each heavy subunit. Although MPO exhibits unusual spectral and enzymatic properties, the nature of the prosthetic group and its mode of linkage with the apoenzyme have not been determined. In an earlier report (K.L. Taylor, J. Pohl, and J.M. Kinkade, Jr. (1992) J. Biol. Chem. 267, 25282-25288), characterization of the autolytic cleavage of MPO led to the proposal that the prosthetic group was covalently linked to the apoenzyme via a methionyl sulfonium bond with Met409. In the present study, we have demonstrated that autolytic cleavage of MPO, followed by protease digestion under nonreducing conditions, effects the release of a macrocycle with visible and Raman spectral properties consistent with that of a protoheme IX derivative. Mass spectrometric analysis, in conjunction with metabolic labeling studies and recent X-ray crystallographic data, have led to the structural assignment of this macrocycle as 1,5-dihydroxymethyl-3,8-dimethyl-4-vinyl-2-(2'-methylthio) ethenylporphine-6,7-dipropionic acid-iron complex. Based on the mechanism of methionyl sulfonium bond cleavage, this structure is consistent with our earlier proposal that the MPO prosthetic group is covalently linked to the enzyme via a methionyl sulfonium bond and suggests that this linkage occurs through a peripheral vinyl substituent.