Catalase-Like Antioxidant Activity is Unaltered in Hypochlorous Acid Oxidized Horse Heart Myoglobin

Abstract

Activated neutrophils release myeloperoxidase that produces the potent oxidant hypochlorous acid (HOCl). Exposure of the oxygen transport protein horse heart myoglobin (hhMb) to HOCl inhibits Iron III (Fe(III))-heme reduction by cytochrome b5 to oxygen-binding Iron II (Fe(II))Mb. Pathological concentrations of HOCl yielded myoglobin oxidation products of increased electrophoretic mobility and markedly different UV/Vis absorbance. Mass analysis indicated HOCl caused successive mass increases of 16 a.m.u., consistent serial addition of molecular oxygen to the protein. By contrast, parallel analysis of protein chlorination by quantitative mass spectrometry revealed a comparatively minor increase in the 3-chlorotyrosine/tyrosine ratio. Pre-treatment of hhMb with HOCl affected the peroxidase reaction between the hemoprotein and H2O2 as judged by a HOCl dose-dependent decrease in spin-trapped tyrosyl radical detected by electron paramagnetic resonance (EPR) spectroscopy and the rate constant of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) oxidation. By contrast, Mb catalase-like antioxidant activity remained unchanged under the same conditions. Notably, HOCl-modification of Mb decreased the rate of ferric-to-ferrous Mb reduction by a cytochrome b5 reductase system. Taken together, these data indicate oxidizing HOCl promotes Mb oxidation but not chlorination and that oxidized Mb shows altered Mb peroxidase-like activity and diminished rates of one-electron reduction by cytochrome b5 reductase, possibly affecting oxygen storage and transport however, Mb-catalase-like antioxidant activity remains unchanged.

Nitroxides Mitigate Neutrophil-Mediated Damage to the Myocardium after Experimental Myocardial Infarction in Rats

“…This is supported by previous studies which have shown that HOCl induces modifications of several proteins resulting in loss of function of these proteins. For example, HOCl oxidatively modifies horse heart myoglobin in vitro and results in loss of reduction of myoglobin from the ferric to the ferrous state caused by cytochrome b5 reductase, an event essential for oxygen transport caused by myoglobin [ 9 ]. Furthermore, HOCl and another MPO oxidant—HOSCN—modify low density lipoprotein (LDL), thereby interfering with endothelial vasorelaxation and inducing endothelial dysfunction [ 35 ].…”

“…Oxidising HOCl also modifies thiol residues and many redox sensitive amino acids (cysteine, tyrosine, and methionine). For example, HOCl induces the chlorination of tyrosine residues, producing the biomarker 3-chlorotyrosine, which is used for quantification of HOCl-induced protein damage [ 8 , 9 ].…”

Lessons from the post-genomic era: Globin diversity beyond oxygen binding and transport

“…Besides NO, Mb also directly regulates ROS homeostasis, acting as both antioxidant and pro-oxidant. Mb exhibits catalase- and peroxidase-like activities thereby neutralizing the ROS-generating H 2 O 2 while itself being converted from ferric-heme to ferryl-heme Mb [ [167] , [168] , [169] , [170] ]. The role of Mb in cardiac antioxidant defense is observed in Mb knockout mice that show poor adaptation to oxidative challenge, including higher ROS in the heart upon cardiac I/R injury and slow recovery from ischemic insult [ 171 ].…”

Strategies of Pathogens to Escape from NO-Based Host Defense

“…In addition, mice infected with T. cruzi and exposed to NO donors are more protected against trypomastigotes compared to the control group [ 196 , 197 ]. Lastly, NO promotes splenocyte apoptosis during the acute phase of T. cruzi infection in mice [ 198 , 199 ] and modulates parasite cell entry, thus contributing to the pathogenesis of Chagas cardiomyopathy [ 188 , 200 , 201 , 202 ].…”