Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation

Ras proteins cycle between GDP-bound and GTP-bound states to modulate a diverse array of cellular growth processes. In this study, we have elucidated a mechanism by which nitric oxide, in the presence of oxygen (NO/O-2), regulates Ras activity. We show that treatment of Ras with NO/O-2 causes conversion of Ras-bound GDP into a free 463.3 Da nucleotide-nitration product. Mass and UV/visible spectroscopic analyses suggest that this nitration product is 5-guanidino-4-nitroimidazole diphosphate (NIm-DP), a degradation product of 5-nitro-GDP. These results indicate that NO/O-2 mediates Ras guanine nucleotide exchange (GNE) by conversion of Ras-bound GDP into an unstable 5-nitro-GDP. 5-Nitro-GDP can be produced by radical-based reaction of the GDP guanine base with nitrogen dioxide ((NO2)-N-.). We also provide evidence that the Ras Phe(28) side-chain plays a key role in the formation of a NO/O-2-induced Ras 5-nitro-GDP product. We previously proposed a mechanism of NO/O-2-mediated Ras GNE, in which (NO2)-N-., formed by the reaction of NO with O-2, generates a Ras Cys(118) thiyl radical (Ras-S-118) intermediate. In the present study, we provide evidence for a radical-based mechanism of NO/O-2-mediated Ras GNE. According to this mechanism, reaction of NO with O-2 produces (NO2)-N-.. (NO2)-N-. then reacts with Ras to produce Ras-S-118., which withdraws an electron from the Ras-bound guanine nucleotide base to produce a guanine nucleotide diphosphate cation radical (G(.+)-DP) via the Phe(28) side-chain. G(.+)-DP is subsequently converted to a neutral radical, and can react with another (NO2)-N-. to produce 5-nitro-GDP. This radical-based reaction process disrupts key binding interactions between Ras and the guanine base, resulting in release of GDP from Ras and its conversion to free 5-nitro-GDP. This mechanism is likely to be common to other NKCD motif-containing Ras superfamily GTPases, as NO/O-2 also facilitates GNE on the redox-active Rap1A and Rab3A GTPases. (C) 2005 Elsevier Ltd. All rights reserved.