Reaction Intermediates and Molecular Mechanism of Peroxynitrite Activation by NO Synthases

Abstract

The activation of the peroxynitrite anion (PN) by hemoproteins, which leads to its detoxification or, on the contrary to the enhancement of its cytotoxic activity, is a reaction of physiological importance that is still poorly understood. It has been known for some years that the reaction of hemoproteins, notably cytochrome P450, with PN leads to the buildup of an intermediate species with a Soret band at~435 nm (I435). The nature of this intermediate is, however, debated. On the one hand, I435 has been presented as a compound II species that can be photoactivated to compound I. A competing alternative involves the assignment of I435 to a ferric-nitrosyl species. Similar to cytochromes P450, the buildup of I435 occurs in nitric oxide synthases (NOSs) upon their reaction with excess PN. Interestingly, the NOS isoforms vary in their capacity to detoxify/activate PN, although they all show the buildup of I435. To better understand PN activation/detoxification by heme proteins, a definitive assignment of I435 is needed. Here we used a combination of fine kinetic analysis under specific conditions (pH, PN concentrations, and PN/NOSs ratios) to probe the formation of I435. These studies revealed that I435 is not formed upon homolytic cleavage of the O-O bond of PN, but instead arises from side reactions associated with excess PN. Characterization of I435 by resonance Raman spectroscopy allowed its identification as a ferric iron-nitrosyl complex. Our study indicates that the model used so far to depict PN interactions with hemo-thiolate proteins, i.e., leading to the formation and accumulation of compound II, needs to be reconsidered.

Decreased Expression and Uncoupling of Endothelial Nitric Oxide Synthase in the Cerebral Cortex of Rats with Thioacetamide-Induced Acute Liver Failure

“…As mentioned earlier, the uncoupling of eNOS in ALF-affected rat brain can be reconciled with, and is a likely contributor to, not only to the accumulation of ROS, but also to a number of subsequent pathogenic events. An ammonia-induced increase in ROS formation, including O 2 − [8], promotes a marked reduction in central and peripheral NO bioavailability by rapid reaction with NO, leading to the formation of highly toxic and short-lived peroxynitrite anion [64,65]. Considerable evidence from in vivo and in vitro studies implicates, directly or indirectly, the role of tyrosine nitration in the evolution of HE symptoms, mainly brain edema [5,[66][67][68].…”

Catalytic Mechanism of Aromatic Nitration by Cytochrome P450 TxtE: Involvement of a Ferric-Peroxynitrite Intermediate

“…Another heme-dependent enzyme, namely nitric oxide synthase (NOS), has also been shown to activate peroxynitrite. , However, in the NOS enzymes, addition of excess peroxynitrite led to the production of the ferrous-nitrosyl species instead. A similar observation was made for P450 BM3 where excess peroxynitrite (ONOO – ) yielded iron­(II)-NO products rather than the anticipated CpdI or CpdII products .…”

Enzymatic Degradation of DNA Probed by In Situ X-ray Scattering

“…Furthermore, the intensity profiles exhibit three clear isosbestic points at q = 0.32, 0.54, and 2.2 nm –1 , where the measured intensity from the Pro-SNA-DNase I system is invariant during the course of the reaction. Such isosbestic points are indicative of a two-state system. − That is, the Pro-SNA composition of the solution at any given time is likely a linear combination of populations of intact Pro-SNA (state A, t = 0) and the final degraded Pro-SNA (state B, t = 6 h). If true, the measured intensity profile I t ( q ) at any intermediate time point in Figure A is Here, I A and I B are the scattered intensities from the pure states A and B, respectively, and α­( t ) is the fraction of Pro-SNA that are in the intact form (state A) at a given time t .…”