Deconstruction of Activity-Dependent Covalent Modification of Heme in Human Neutrophil Myeloperoxidase by Multistage Mass Spectrometry (MS⁴)

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.

Pharmacokinetics and Disposition of the Thiouracil Derivative PF-06282999, an Orally Bioavailable, Irreversible Inactivator of Myeloperoxidase Enzyme, Across Animals and Humans

“…The mode of inhibition is similar to the one described with the recently reported 2-thioxanthine class of MPO inactivators (e.g., compound TX5 in Fig. 1) (Tidén et al, 2011;Geoghegan et al, 2012).…”

Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine: biochemical and X-ray structural characterization

“…In this mechanism, the chemical reactivity of 2TX results from its peculiar interaction with its enzyme binding site (conserved aromatic site), whereas the free molecule in solution can be considered hardly reactive in its major oxothione tautomer (Figure 1a). This molecular mechanism is very different from the one proposed for the inhibition of Myelopexoxidase (MPO, an enzyme involved in oxidative stress during inflammation) by 2TX and its N3-alkylated-derivatives (63,64). In this case, the inhibition leads to the oxidation of the drug resulting in a covalent adduct between the sulfur atom of 2TX and the enzyme heme prosthestic group by following a complex radical chemistry used naturally by MPO to oxidize several of its substrates (here and differently from our case, 2TX-derivatives mimic true substrates).…”

Cardiac Phosphoproteomics during Remote Ischemic Preconditioning: A Role for the Sarcomeric Z-Disk Proteins

“…Cardiac proteomic studies provide insight into changes in total protein expression during cardiac disease and for the identification and localization of posttranslational modifications with the hope that this technique will also help to identify markers of cardiac disease as well as novel therapeutic targets [36–41]. Two-dimensional gel electrophoresis has been used to visualize the proteome profile in the border zone of the early stage postinfarct [42] and proteins that were differentially expressed in the ischemically reperfused heart compared with the control and ischemically preconditioned rat hearts [43].…”