Mechanism of the nitrosation of thiols and amines by oxygenated ^•NO solutions: The nature of the nitrosating intermediates

The nitrosation of various thiols and morpholine by oxygenated ^•NO solutions at physiological pH was investigated. The formation rates and the yields of the nitroso compounds were determined using the stopped-flow technique. The stoichiometry of this process has been determined, and is given by 4^•NO + O₂ + 2RSH/2RR′NH → 2RSNO/2RR¹NNO + 2NO₂^- + 2H⁺. Kinetic studies show that the rate law is -d[O₂]/dt = k₁[^•NO]²[O₂] with k₁ = (2.54 ± 0.26) × 10⁶ M^-2 s^-1 and -d[^•NO]/dt = 4k₁[^•NO]²[O₂] with 4k₁ = (1.17 ± 0.12) × 10⁷ M^-2 s^-1, independent of the kind of substrate present. The kinetic results are identical to those obtained for the autoxidation of ^•NO, indicating that the rate of the autoxidation of ^•NO is unaffected by the presence of thiols and amines. The nitrosation by ^•NO takes place only in the presence of oxygen, and therefore the rate of the formation of S-nitrosothiols from thiols and oxygenated ^•NO solution is relatively slow in biological systems. Under physiological conditions where [^•NO] < 1 μM and [O₂] < 200 μM, the half-life of the nitrosation process exceeds 7 min. Therefore, this is an unlikely biosynthetic pathway for the formation of S-nitrosothiols. As such, S-nitrosothiols cannot serve as carrier molecules of ^•NO in vivo. The rate-determining step of the nitrosation of thiols and amines by oxygenated ^•NO solution is the formation of ONOONO (or ONONO₂ or O₂NNO₂), which is the precursor of ^•NO₂ and N₂O₃. The stoichiometry of the nitrosation process suggests that ^•NO₂ and/or N₂O₃ are the reactive species. We have demonstrated that ^•NO₂ initiates the nitrosation process unless it is scavenged faster by ^•NO to form N₂O₃. The latter entity is also capable of directly nitrosating thiols and amines with rate constants exceeding 6 × 10⁷ M^-1 s^-1.