Reaction Intermediates and Molecular Mechanism of Peroxynitrite Activation by NO Synthases.

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.