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 + O 2 + 2RSH/2RR‘NH → 2RSNO/2RR’NNO + 2NO 2 - + 2H + . Kinetic studies show that the rate law is − d [O 2 ]/d t = k 1 [ • NO] 2 [O 2 ] with k 1 = (2.54 ± 0.26) × 10 6 M - 2 s - 1 and − d [ • NO]/d t = 4 k 1 [ • NO] 2 [O 2 ] with 4 k 1 = (1.17 ± 0.12) × 10 7 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 [O 2 ] < 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 ONONO 2 or O 2 NNO 2 ), which is the precursor of • NO 2 and N 2 O 3 . The stoichiometry of the nitrosation process suggests that • NO 2 and/or N 2 O 3 are the reactive species. We have demonstrated that • NO 2 initiates the nitrosation process unless it is scavenged faster by • NO to form N 2 O 3 . The latter entity is also capable of directly nitrosating thiols and amines with rate constants exceeding 6 × 10 7 M - 1 s - 1 .