Protein tyrosine nitration and peroxynitrite

In a recent paper by Pfeiffer et al. (1), the authors argue against an essential role for peroxynitrite in the nitration of protein tyrosine residues in inflammatory diseases. Central to this argument is their finding, using macrophages isolated from mice, that O2 and NO were not detectable simultaneously. That is, cellular O2 release as measured by the reduction of ferricytochrome c appeared to cease 8 hours after stimulation (their Fig. 2), which is the time at which NO formation began to be detected (their Fig. 1). Because peroxynitrite is formed by the reaction of O2 with NO, it was reasoned that the tyrosine nitration observable after 12 hours came from some other source. However, it is almost certain that the apparent cessation of O2 release is an artifact caused by the failure to inhibit NO synthesis when using the cytochrome c assay. Because the reaction of O2 with NO is much faster than that with ferricytochrome c (see below), the presence of even small concentrations of NO will make O2 release ‘invisible’ using this approach. A comparison of the competing rates of reaction emphasizes how important it is to suppress NO synthesis when trying to detect O2 with cytochrome c. The rate constants for O2 with NO and ferricytochrome c are 6.7 10 M s 1 (2) and 1.1 10 M s 1 (3), respectively. Multiplying these by a typical NO concentration of 1 M in macrophage cultures and a concentration of (acetylated) cytochrome c of 10 M in the cited assay (4), one finds that the reaction rate with NO is some 600 times faster than that with ferricytochrome c. Accordingly, the failure to observe O2 using this approach, once induction of NO synthesis has occurred, is predictable. In addition, reduced cytochrome c is reoxidized by peroxynitrite, further diminishing its effectiveness in measuring superoxide (5).