Myeloperoxidase interaction with peroxynitrite: chloride deficiency and heme depletion

Abstract Myeloperoxidase (MPO) is a hemoprotein involved in the leukocyte-mediated defense mechanism and uses hydrogen peroxide (H 2 O 2 ) and chloride (Cl − ) to produce hypochlorous acid. In human saliva and in hypochloremic alkalosis syndrome occurring in breast-fed infants, the MPO–H 2 O 2 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 H 2 O 2 caused immediate formation of Compound II, followed by MPO heme depletion, a process that occurred independent of ONOO − concentration. Peroxynitrite/H 2 O 2 -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.