Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group

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Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 94%

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Disruption of the Aspartate to Heme Ester Linkage in Human Myeloperoxidase

Zederbauer

Furtmüller

Bellei

et al. 2007

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“…Although crystal structures for other family members have not been solved, modeling of homologous domains, NMR, mass spectrometry, and peptide analysis support the presence of the same two ester linkages in LPO, EPO, and TPO (11, 70 -74). Ester bonds account for the 10 nm shift in the Soret band of LPO, EPO, and TPO relative to horseradish peroxidase, and the presence of the third covalent bond in MPO causes the red shift of the Soret band of oxidized MPO ( max ϭ 430 nm) relative to the spectral peak for the other family members ( max ϭ 412 nm) (73,75,76). The peculiar sulfonium linkage in MPO likely contributes as well to its unique facility to oxidize Cl Ϫ to HOCl at physiologic pH.…”

Section: Discussionmentioning

confidence: 99%

Impact of Two Novel Mutations on the Structure and Function of Human Myeloperoxidase

Goedken

McCormick

Leidal

et al. 2007

The heme protein myeloperoxidase (MPO) contributes critically to O 2 -dependent neutrophil antimicrobial activity. Two Japanese adults were identified with inherited MPO deficiency because of mutations at Arg-499 or Gly-501, conserved residues near the proximal histidine in the heme pocket. Because of the proximity of these residues to a critical histidine in the heme pocket, we examined the biosynthesis, function, and spectral properties of the peroxidase stably expressed in human embryonic kidney cells. Biosynthesis of normal MPO by human embryonic kidney cells faithfully mirrored events previously identified in cells expressing endogenous MPO. Mutant apopro-MPO was 90 kDa and interacted normally with the molecular chaperones ERp57, calreticulin, and calnexin in the endoplasmic reticulum. However, mutant precursors were not proteolytically processed into subunits of MPO, although secretion of the unprocessed precursors occurred normally. Although ␦-[14 C]aminolevulinic acid incorporation demonstrated formation of pro-MPO in both mutants, neither protein was enzymatically active. The Soret band for each mutant was shifted from the normal 430 to ϳ412 nm, confirming that heme was incorporated but suggesting that the number of covalent bonds or other structural aspects of the heme pocket were disrupted by the mutations. These studies demonstrate that despite heme incorporation, mutations in the heme environs compromised the oxidizing potential of MPO.

“…Human TPO has been shown to contain a valine at this position (18). In a recent study (19), we have mutated the Met 243 of MPO into a glutamine to create an LPO-like protein. This mutant MPO is spectroscopically very similar to LPO, and we concluded that Met 243 is responsible for the spectral characteristics of MPO.…”

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confidence: 99%

The Sulfonium Ion Linkage in Myeloperoxidase

Kooter¹,

Moguilevsky²,

Bollen³

et al. 1999

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The heme group of myeloperoxidase is covalently linked via two ester bonds to the protein and a unique sulfonium ion linkage involving Met 243 . Mutation of Met 243 into Thr, Gln, and Val, which are the corresponding residues of eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively, and into Cys was performed. The Soret band in the optical absorbance spectrum in the oxidized mutants is now found at approximately 411 nm. Both the pyridine hemochrome spectra and resonance Raman spectra of the mutants are affected by the mutation. In the Met 243 mutants the affinity for chloride has decreased 100-fold. All mutants have lost their chlorination activity, except for the M243T mutant, which still has 15% activity left. By Fourier transform infared difference spectroscopy it was possible to specifically detect the 13 CD 3 -labeled methionyl sulfonium ion linkage. We conclude that the sulfonium ion linkage serves two roles. First, it serves as an electron-withdrawing substituent via its positive charge, and, second, together with its neighboring residue Glu 242 , it appears to be responsible for the lower symmetry of the heme group and distortion from the planar conformation normally seen in heme-containing proteins.In the family of mammalian peroxidases, myeloperoxidase (MPO) 1 is an extraordinary peroxidase. First of all, the enzyme is the only mammalian peroxidase known to peroxidize chloride to hypochlorous acid at a substantial rate. Secondly, MPO differs in its spectroscopic characteristics by its unusual redshifted Soret band in the optical absorbance as well in its pyridine hemochrome spectrum, its complicated resonance Raman spectrum, and its inverted sign pattern of the Soret band in the MCD spectrum (1-6). Those differences have been attributed to the special nature or structure of the heme group in MPO. Because this heme is covalently bound to the protein, characterization of this chromophore has been difficult. Based on different spectroscopic techniques, a formyl-containing heme a, a chlorin, and a heme b prosthetic group have been proposed in the past (3,5,(7)(8)(9).Although the enzyme differs in spectroscopic and catalytic properties, the homology between MPO and the other mammalian peroxidases is high. MPO shares respectively 70, 61, and 47% identical residues with eosinophil peroxidase (EPO), lactoperoxidase (LPO), and thyroid peroxidase (TPO) (10 -12), and an even higher homology can be found among the residues in the active site. MPO is the only mammalian peroxidase for which a crystal structure is known, at 2.3 Å resolution (13). The structural data for human MPO suggested that three heme substituents form covalent bonds with amino acid side chains in the protein. Two ester bonds were claimed to be present, between modified methyl groups on pyrrole rings A and C and the amino acids Glu 242 and Asp 94 . In a recent study, we have provided the first direct evidence by FTIR difference spectroscopy that ester bonds link the heme groups in all mammalian peroxidases via the conser...