Myeloperoxidase (MPO) is a hemoprotein involved in the leukocyte-mediated defense mechanism and uses hydrogen peroxide (H2O2) and chloride (Cl-) to produce hypochlorous acid. In human saliva and in hypochloremic alkalosis syndrome occurring in breast-fed infants, the MPO-H2O2 system functions in a lower Cl- concentration (10-70 mM) compared to plasma levels (100 mM) as part of the antibacterial defense system. The impact of low Cl- concentration and exposure to high peroxynitrite (ONOO-) synthesized from cigarette smoke or oxidative stress on MPO function is still unexplored. Rapid mixing of ONOO- and MPO caused immediate formation of a transient intermediate MPO Compound II, which then decayed to MPO-Fe(III). Double mixing of MPO with ONOO- followed by H2O2 caused immediate formation of Compound II, followed by MPO heme depletion, a process that occurred independent of ONOO- concentration. Peroxynitrite/H2O2-mediated MPO heme depletion was confirmed by HPLC analysis, and in-gel heme staining showing 60-70% less heme content compared to the control. A nonreducing denaturing SDS-PAGE showed no fragmentation or degradation of protein. Myeloperoxidase heme loss was completely prevented by preincubation of MPO with saturating amounts of Cl-. Chloride binding to the active site of MPO constrains ONOO- binding by filling the space directly above the heme moiety or by causing a protein conformational change that constricts the distal heme pocket, thus preventing ONOO- from binding to MPO heme iron. Peroxynitrite interaction with MPO may serve as a novel mechanism for modulating MPO catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.
|Number of pages||9|
|Journal||Free Radical Biology and Medicine|
|State||Published - Aug 15 2009|
- Free radicals
- Hydrogen peroxide
- Hypohalous acid
- Mammalian peroxidase