Optical spectrum of myeloperoxidase

Abstract

The optical spectrum of reduced myeloperoxidase (EC 1.11.1.7) displays an unusual red shift of the Soret band which is at 472 nm and the a-band which is at 636 nm. The spectral properties of myeloperoxidase can be modified by means of acid treatment. Upon short exposure to acid (pH 1.7) the red-shifted optical absorption spectrum of the reduced enzyme (A,,, at 472 nm) was blue-shifted (Ama at 448 nm) but the spectrum of the reduced state could be restored by increasing the pH. By contrast, the resonance Raman spectra of both the oxidized and reduced enzyme are essentially the same at both pH 1.7 and pH 7.0. This shows that the optical spectrum and the resonance Raman spectrum are not directly correlated, which we interpret to indicate that the reversible effects of lower pH primarily affect the excited-state energy levels of the macrocycle. The EPR spectrum of the oxidized enzyme showed a reversible conversion from a high-spin rhombic spectrum (8, = 6.7, g, = 5.2) at neutral pH into a more axial high-spin spectrum (gx = g, = 5.8) at low pH.Upon prolonged exposure to acid (20 min) optical absorbance spectra, EPR spectra, resonance Raman spectra and the chlorinating activity were irreversibly affected. We propose that a negatively charged protonatable residue in the proximity of a pyrrole nucleus of the haem group is present that imposes the red shift in the optical absorption spectrum. This is consistent with the available X-ray structure data.Myeloperoxidase is present in large amounts in polymorphonuclear neutrophils and plays an important role in the antimicrobial activity. The enzyme catalyzes the H,O,-dependent peroxidation of chloride to hypochlorous acid, which is a bactericidal agent [l, 21. Myeloperoxidase has also been shown to react very rapidly with peroxynitrite and a new role for the enzyme as a scavenger for peroxynitrite has been proposed [3].The optical absorption spectrum of the reduced enzyme is unique because the Soret band (472nm) and the a-band (636 nm) are red-shifted compared with other haem proteins [4]. The chemical nature of the prosthetic group of myeloperoxidase, which is a haem group, has, however, not been identified, partly due to the fact that the haem group is not readily extractable. This is possibly the result of a covalent linkage between the prosthetic group and the protein [5-71. The crystal structure has been determined at 0.3-nm resolution [S], however, the exact nature of the prosthetic group could not be established from the current X-ray data. It has been suggested that the chromophore is a porphyrin with electronwithdrawing substituents because pyridine haemochrome spectra and the spectra of the enzyme treated with SDS or acid have a strong similarity with that of the haem a spectra of the corresponding form of cytochrome c oxidase [9-131.

Difference Fourier Transform Infrared Evidence for Ester Bonds Linking the Heme Group in Myeloperoxidase, Lactoperoxidase, and Eosinophil Peroxidase

“…This result demonstrates conclusively that the second set of features observed arise from an ester group derived from Asp94. Our results are not consistent with the earlier proposal that glutamate 242 and aspartate 94 influence the heme group via electrostatic effects due to the negatively charged carboxylate residues. , Instead, our data demonstrate that the two carboxylic acid residues are covalently attached to the hydroxylated heme group by esterification.

2 Reduced−oxidized FTIR difference spectra of wild-type MPO, Glu242Gln mutant MPO (×2), and Asp94Val mutant MPO (×3).

…”

Characterization of Mitochondrial and Extra-mitochondrial Oxygen Consuming Reactions in Human Hematopoietic Stem Cells

“…This suggested the presence of a significant amount of extramitochondrial cytochromes (see below). In addition, the differential absorbance at 470 -475 nm clearly indicated the presence of myeloperoxidase (which specifically absorbs in that region (24)). …”

Biochemical Evidence for Heme Linkage through Esters with Asp-93 and Glu-241 in Human Eosinophil Peroxidase

“…Unlike the two acidic residues, this methionine has no obvious equivalent in LPO or EPO (11,25). It has also been suggested that Asp-94 and Glu-242 exist as protonatable residues capable of influencing the absorption spectrum of the heme group (27,28). In short, however, after many years of investigation with mixed results, linkage of the heme group through two esters seems to be accepted for LPO and MPO, although for LPO, the residue that corresponds to Asp-94 in MPO has not been unambiguously identified (24).…”