Abstract
Hydrogen sulfide (H(2)S) and nitric oxide (NO) are two relevant signal molecules that can affect protein function throughout post-translational modifications (PTMs) such as persulfidation, S-nitrosation, metal-nitrosylation, and nitration. Lipoxygenases (LOXs) are a group of non-heme iron enzymes involved in a wide range of plant physiological functions including seed germination, plant growth and development, and fruit ripening and senescence. Likewise, LOXs are also involved in the mechanisms of response to diverse environmental stresses. Using purified soybean (Glycine max L.) lipoxygenase type 1 (LOX 1) and nitrosocysteine (CysNO) and sodium hydrosulfide (NaHS) as NO and H(2)S donors, respectively, the present study reveals that both compounds negatively affect LOX activity, suggesting that S-nitrosation and persulfidation are involved. Mass spectrometric analysis of nitrated soybean LOX 1 using a peroxynitrite (ONOO(-)) donor enabled us to identify that, among the thirty-five tyrosine residues present in this enzyme, only Y214 was exclusively nitrated by ONOO(-). The nitration of Y214 seems to affect its interaction with W500, a residue involved in the substrate binding site. The analysis of the structure 3PZW demonstrates the existence of several tunnels that directly communicate the surface of the protein with different internal cysteines, thus making feasible their potential persulfidation, especially C429 and C127. On the other hand, the CysNO molecule, which is hydrophilic and bulkier than H(2)S, can somehow be accommodated throughout the tunnel until it reaches C127, thus facilitating its nitrosation. Overall, a large number of potential persulfidation targets and the ease by which H(2)S can reach them through the diffuse tunneling network could be behind their efficient inhibition.