Direct detection of the oxygen rebound intermediates, ferryl Mb and NO2, in the reaction of metmyoglobin with peroxynitrite.
Oxygenated hemoproteins are known to react rapidly with nitric oxide (NO) to produce peroxynitrite (PN) at the heme site. This process could lead either to attenuation of the effects of NO or to nitrosative protein damage. Peroxynitrite is a powerful nitrating and oxidizing agent that has been implicated in a variety of cell injuries. Accordingly, it is important to delineate the nature and variety of reaction mechanisms of PN reactions with heme proteins. Here, we present direct evidence that ferrylMb and NO(2) are both produced during the reaction of PN and metmyoglobin (metMb). Kinetic evidence indicates that these products evolve from initial formation of a caged radical intermediate [Fe(IV)=O *NO(2)]. This caged pair reacts mainly via internal return with a rate constant k(r) to form metMb and nitrate in an oxygen rebound scenario. Detectable amounts of ferrylMb are observed by stopped-flow spectrophotometry, appearing at a rate consistent with the rate, k(obs), of heme-mediated PN decomposition. Freely diffusing NO(2), which is liberated concomitantly from the radical pair (k(e)), preferentially nitrates Tyr103 in horse heart myoglobin. The ratio of the rates of in-cage rebound and cage escape, k(r)/k(e), was found to be approximately 10 by examining the nitration yields of fluorescein, an external NO(2) trap. This rebound/escape model for the metMb/PN interaction is analogous to the behavior of alkyl hyponitrites and the well-studied geminate recombination processes of deoxymyoglobin with O(2), CO, and NO. The scenario is also similar to the stepwise events of substrate hydroxylation by cytochrome P450 and other oxygenases. It is likely, therefore, that the reaction of metMb with ONOO(-) and that of oxyMb with NO proceed through the same [Fe(IV)=O *NO(2)] caged radical intermediate and lead to similar outcomes. The results indicate that while oxyMb may reduce the concentration of intracellular NO, it would not eliminate the formation of NO(2) as a decomposition product of peroxynitrite.
Su
Jia
J
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Groves
John T
JT
D000818:Animals; D006736:Horses; D007700:Kinetics; D008786:Metmyoglobin / Q000032:analysis* / Q000378:metabolism; D008958:Models, Molecular; D009211:Myoglobin / Q000378:metabolism; D009585:Nitrogen Dioxide / Q000032:analysis* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D009211:Myoglobin; C028048:ferrylmyoglobin; D008786:Metmyoglobin; D030421:Peroxynitrous Acid; D014443:Tyrosine; D009585:Nitrogen Dioxide; D010100:Oxygen
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