However a direct effect of ROS

However, a direct effect of ROS on GSNOR has also been described. Inhibition of GSNOR by ROS has been demonstrated for yeast [44], Arabidopsis[36], and Baccaurea ramiflora (Burmese grape) [45] suggesting that this could be a general mechanism of crosstalk between ROS and ·No signaling. Interestingly, ROS target different cysteine residues in comparison to SNO-dependent inhibition of GSNOR. Treatment with 10 µM H2O2 resulted in 15% inhibition, which further decreased to 35% in presence of 1 mM H2O2. Light scattering analysis demonstrated that under oxidizing as well as under reducing conditions, AtGSNOR appeared as homodimer, concluding that no drastic changes of the native structure of AtGSNOR, such as monomerization, are caused by oxidative conditions. Changes in the mobility could be observed for H2O2-treated AtGSNOR in a non-reducing SDS PAGE analysis, which could be reversed under reducing conditions. These slight structural changes might at least partly be responsible for the H2O2-induced inhibition of AtGSNOR. Three cysteine residues (Cys47, Cys177, Cys271) are located in the substrate binding site (Fig. 2), where Cys47 and Cys177 are involved in coordinating the catalytic Zn2+. Especially these two cysteine residues are sensitive to oxidation, whereas Cys271 localized in the NAD + cofactor binding site is resistant to H2O2 induced Closantel Sodium mg [36]. The importance of Cys47 and Cys177 for the catalytic activity of GSNOR is also demonstrated by the corresponding serine mutants, which show up to 100-fold less specific activity in comparison to WT GSNOR. The reduction of GSNOR activity after H2O2 treatment is a least partly due to loss of the catalytic Zn2+. A H2O2 concentration-dependent Zn2+-release of GSNOR suggested a correlation between oxidation of cysteine residues, loss of activity and Zn2+-release. Moreover, excess of external Zn2+ can prevent H2O2-caused inhibition of GSNOR activity confirming that H2O2-dependent oxidation of cysteine residues results in zinc release and loss of GSNOR activity. In the structurally related yeast alcohol dehydrogenase 1 (ADH1) H2O2-treatment resulted in oxidation of the cysteine residues corresponding to Cys47 (Cys43) and Cys177 (Cys153). Similar as for AtGSNOR, oxidative modifications were accompanied by Zn2+-release in yeast ADH1 [46]. Among all cysteine residues in yeast ADH1 Cys43 is most susceptible to H2O2-caused oxidations and the major oxidation products of Cys43 were the corresponding sulfenic and sulfonic acids. These oxidations might be the cause for the H2O2-dependent inactivation of the yeast ADH1. Additionally, formation of disulfide bridges between Cys43-Cys153 in the catalytic domain, Cys103-Cys111 in the non-catalytic Zn2+ center and Cys276-Cys277 has been observed. However, the function of the disulfide formation is not clear. Since the majority of enzyme activity loss could not be restored by reducing condition, the formation of disulfide bond is not the major oxidation pathway leading to the activity loss. In AtGSNOR neither intramolecular nor intermolecular disulfide bond formation has been observed after H2O2 treatment. Most likely oxidation of the Zn2+ coordinating Cys43 to sulfenic and sulfonic acid might be the cause for the H2O2-dependent inactivation of the yeast ADH1. Approximately 1.2 zinc ions and 3 thiols were lost when the enzyme was completely inactivated. Oxidative modification of Cys103 and Cys111, both coordinating the structural Zn2+, did not correlate with enzyme activity loss and is therefore is not the major contributor to enzyme inactivation. Recently, calmodulin (CaM)-dependent inhibition of Arabidopsis GSNOR was reported [28]. Calmodulin is an important multifunctional Ca2+ sensor protein in plants and the presented results suggest that AtCaM1 and AtCaM4 serve as signals in plant salt resistance by promoting NO accumulation through the binding and inhibition of GSNOR. However, if CaM binding and redox-modifications of GSNOR are somehow connected or do interfering is not known.