Myeloperoxidase Interaction with Peroxynitrite: Chloride Deficiency and Heme Depletion

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 hypochloremic alkalosis syndrome occurring in breast fed infants, the MPO-H2O2 system functions in 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 occured 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 non-reducing denaturing SDS PAGE showed no fragmentation or degradation of protein. Myeloperoxidase heme loss was completely prevented by pre-incubation of MPO with saturated 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.