Kinetics of the anaerobic reaction of nitric oxide with cysteine, glutathione and cysteine-containing proteins: implications for in vivo S-nitrosation

Abstract

A study is made of the kinetics of the anaerobic reaction of nitric oxide (˙NO) with cysteine and glutathione in relation to its potential physiological importance for the S-nitrosation of cysteine-containing peptides and proteins. The kinetics of the reaction with cysteine (the basic reagent unit) is studied most extensively and it is found that the rate constant is directly proportional to the degree of ionization (α), k = 0.37 × 10³α M⁻¹ s⁻¹, offering clear proof for a mechanism based on electrophilic attack of ˙NO on thiolate anions. The rate constant for glutathione is considerably lower than for cysteine at identical pH, which can be attributed to its higher pK_a; steric effects do not appear to affect the reactivity of glutathione significantly. On the basis of the rate equations obtained and of similar data for bovine serum albumin and metallothionein-1 a number of calculations were performed with the aim of determining the relative importance of the reaction of ˙NO with O₂vs. the direct reaction of ˙NO with peptide and protein thiols under in vivo conditions. The results clearly show that in cells that as a rule contain an appreciable concentration of glutathione the autoxidation of ˙NO and thus the reaction of higher oxides of nitrogen (˙NO₂, N₂O₃) with thiol groups in peptides and proteins does not play a role of any significance with respect to the formation of S-nitrosothiols, as the direct reaction of ˙NO with the thiolate group in glutathione leading to the formation of GS–˙NO⁻ is much faster than the reaction of ˙NO with O₂. The difference in reactivity is less pronounced in the case of bovine serum albumin, but again the electrophilic attack clearly is more important than the autoxidation at most physiological ˙NO concentrations. Direct electrophilic attack of ˙NO on metallothionein is of no practical significance, as the process is very much slower than the attack on glutathione.