Abstract

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.

Links

Publisher Full Text

Authors

Geoghegan KF, Varghese AH, Feng X, Bessire AJ, Conboy JJ, Ruggeri RB, Ahn K, Spath SN, Filippov SV, Conrad SJ, Carpino PA, Guimarães CR, Vajdos FF

Institution

Pfizer Worldwide Research, Groton, Connecticut 06340, United States. kieran.f.geoghegan@pfizer.com

Source

Biochemistry 51:10 2012 Mar 13 pg 2065-77

MeSH

Amino Acid Sequence
Catalytic Domain
Chromatography, Liquid
Crystallography, X-Ray
Heme
Humans
Models, Chemical
Models, Molecular
Molecular Sequence Data
Molecular Weight
Neutrophils
Oxidation-Reduction
Peptide Fragments
Peptide Mapping
Peroxidase
Tandem Mass Spectrometry

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

22352991