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 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.
Galijasevic
Semira
S
Obstetrics and Gynecology, The C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Maitra
Dhiman
D
Lu
Tun
T
Sliskovic
Inga
I
Abdulhamid
Ibrahim
I
Abu-Soud
Husam M
HM
D000471:Alkalosis / Q000139:chemically induced; D000494:Allosteric Regulation; D001942:Breast Feeding / Q000009:adverse effects; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007111:Immunity, Cellular; D066298:In Vitro Techniques; D007223:Infant; D007962:Leukocytes / Q000201:enzymology; D018384:Oxidative Stress; D009195:Peroxidase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000009:adverse effects / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012907:Smoking / Q000009:adverse effects / Q000378:metabolism
D002712:Chlorides; D030421:Peroxynitrous Acid; D006418:Heme; D009195:Peroxidase