HCN regulates cellular processes through posttranslational modification of proteins by S-cyanylation.

Hydrogen cyanide (HCN) is coproduced with ethylene in plant cells and primarily enzymatically detoxified by the mitochondrial ß-cyanoalanine synthase (CAS-C1). Permanent or transient depletion of CAS-C1 activity in Arabidopsis results in physiological alterations in the plant that suggest that HCN acts as a gasotransmitter molecule. Label-free quantitative proteomic analysis of enriched mitochondrial samples isolated from the wild type and cas-c1 mutant revealed significant changes in protein content, identifying 451 proteins that are absent or less abundant in cas-c1 and 353 proteins that are only present or more abundant in the mutant background. Gene ontology classification of these proteins highlights proteomic changes that explain the root hairless phenotype and the altered immune response observed in the cas-c1 mutant. The mechanism of action of cyanide as a signaling molecule was addressed using two proteomic approaches focused on identifying the S-cyanylation of cysteine as a posttranslational modification of proteins. Both the 2-imino-thiazolidine chemical method and the direct untargeted analysis of proteins using LC-MS/MS identified a set of 163 proteins susceptible to S-cyanylation that included sedoheptulose 1, 7-bisphosphatase (SBPase), the peptidyl-prolyl cis-trans isomerase (CYP20-3) and enolase 2 (ENO2). In vitro analysis of these enzymes showed that this modification in the SBPase Cys74, CYP20-3 Cys259, and ENO2 Cys346 residues affected their enzymatic activity. GO classification and protein-protein interaction cluster analysis showed that S-cyanylation is involved in the regulation of primary metabolic pathways, such as glycolysis, and the Calvin and S-adenosylmethionine cycles.