# Old poisons, new signaling molecules: the case of hydrogen cyanide

Pablo Díaz-Rueda, Laura Morales de los Ríos, Luis C Romero, Irene García

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Pablo Díaz-Rueda, Laura Morales de los Ríos, Luis C Romero, Irene García. Old poisons, new signaling molecules: the case of hydrogen cyanide. Journal of Experimental Botany, 2024, 74, pp.6040 - 6051. 10.1093/jxb/erad317. hal-04837488

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REVIEW PAPER

# Old poisons, new signaling molecules: the case of hydrogen cyanide

Pablo Díaz-Rueda†, Laura Morales de los Ríos†,‡, Luis C. RomeroID and Irene García*

Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), CSIC-Universidad de Sevilla, 41092-Sevilla, Spain

‡ Present address: Institut des Sciences des Plantes de Montpellier (IPSIM), CNRS/INRAE/SupAgro-M/UM2, 34060-Montpellier Cedex 1, France.

† These authors contributed equally to this work.

* Correspondence: irene.garcia@ibvf.csic.es

Received 20 April 2023; Editorial decision 4 August 2023; Accepted 14 August 2023

Editor: Angeles Aroca, Universidad de Sevilla, Spain

## Abstract

The high phenotypic plasticity developed by plants includes rapid responses and adaptations to aggressive or changing environments. To achieve this, they evolved extremely efficient mechanisms of signaling mediated by a wide range of molecules, including small signal molecules. Among them, hydrogen cyanide (HCN) has been largely ignored due to its toxic characteristics. However, not only is it present in living organisms, but it has been shown that it serves several functions in all kingdoms of life. Research using model plants has changed the traditional point of view, and it has been demonstrated that HCN plays a positive role in the plant response to pathogens independently of its toxicity. Indeed, HCN induces a response aimed at protecting the plant from pathogen attack, and the HCN is provided either exogenously (in vitro or by some cyanogenic bacteria species present in the rhizosphere) or endogenously (in reactions involving ethylene, camalexin, or other cyanide-containing compounds). The contribution of different mechanisms to HCN function, including a new post-translational modification of cysteines in proteins, namely S-cyanylation, is discussed here. This work opens up an expanding ‘HCN field’ of research related to plants and other organisms.

**Keywords:** Hydrogen cyanide, immune response, metalloproteins, plant defense, S-cyanylation, signaling.

## Introduction

As sessile organisms, plants are unable to move to avoid adverse conditions. Therefore, they evolved extremely efficient mechanisms for detecting and responding to the wide diversity of biotic and/or abiotic stress conditions to restore cellular homeostasis.

The detection of changes in external conditions acquires special relevance and, concomitantly, the signaling mechanisms triggering responses aimed at restoring homeostasis or adapting their physiology to the new conditions are extremely

efficient. The molecular mechanisms that underlie cell signaling in plants constitute a central topic of research, especially in our current climatic change scenario. Among them, the role of small signal molecules deserves attention because they are able to trigger rapid responses mainly due to their high chemical reactivity.

Hydrogen cyanide (HCN) is a well-known poison used since ancient times for suicides and murders, including mass
killings. Additionally, industrial wastes and leakages of HCN are the origin of environmental pollution and catastrophes. It has been extensively referred to in detective books, where it often appears as a substance with the characteristic odor of bitter almonds. It is a low molecular weight molecule that is highly reactive, soluble in water, and has a low melting point. Due to its reactivity and its abundance in the Earth's earliest atmosphere, the participation of HCN in the origin of ribonucleotides, lipids, and amino acids is more than possible (Patel *et al.*, 2015). Its toxic capacity is mainly due to its ability to form very stable complexes with transition metals of the prosthetic groups of metalloproteins that are essential for their function (Nagahara *et al.*, 1999). The main target of cyanide in living organisms is the mitochondria, where it blocks electron transfer by cytochrome *c* oxidase and thus interrupts mitochondrial oxygenic respiration (Donato *et al.*, 2007), although it also affects photosynthetic enzymes in chloroplasts (Berg and Krogmann, 1975) and the activity of enzymes such as catalase and oxidases (Cheeke, 1995; McMahon *et al.*, 1995).

Therefore, HCN has been considered only as a toxic compound whose presence is rapidly eliminated by detoxifying activities. However, its physicochemical characteristics are similar of those of other low molecular weight signaling molecules, such as nitric oxide (NO) and hydrogen sulfide ($H_2S$), which are toxic at high concentrations and possess, at non-toxic concentrations, a widely demonstrated and accepted signaling role in plants (Delledonne *et al.*, 2001; Astier and Lindermayr, 2012; Paul and Snyder, 2015; Aroca *et al.*, 2018).

## HCN in life kingdoms

HCN has been described to be synthetized within cells of organisms from all kingdoms except *Archaea*. In bacteria and fungi, the amino acid glycine is oxidized and decarboxylated by the membrane-bound flavoenzyme cyanide synthase, giving HCN and $CO_2$ as products (Knowles, 1976, 1988; Blumer and Haas, 2000). In these organisms, HCN and HCN-containing molecules such as cyanogenic glucosides can serve as a nitrogen source for amino acids and other N-containing molecules, but other roles for HCN have been identified or suggested (Fig. 1). Some HCN-emitting soil bacterial strains, especially fluorescent pseudomonads, have important effects against plant diseases and, historically, this effect has been attributed to a direct poisonous effect. However, it has also been shown that these bacteria stimulate plant growth depending on their capacity to emit HCN (Kuzmanovic *et al.*, 2018; Sehrawat *et al.*, 2022; see below). In the context of animal-bacteria interactions, it is interesting to mention the case of *Pseudomonas aeruginosa*, an opportunistic human pathogen affecting the lungs and causing pneumonia. A wide range of *P. aeruginosa* strains produce HCN, which has been shown to be involved in the process of lung tissue colonization by quorum-sensing mechanisms by inducing the production of biofilm components and by competition with other bacterial

species, such as *Staphylococcus aureus*, not only in proximity but also at a distance (Létoffé *et al.*, 2022).

Cyanogenesis in arthropods has also been described. In many cases, arthropods accumulate cyanogenic compounds from the plants they feed on, and they prefer cyanogenic rather than non-cyanogenic plants to feed on (Zagrobelny *et al.*, 2007, 2008), but some arthropods are able to synthesize cyanogenic glucosides *de novo* as well (Siegien and Bogatek, 2006; Zagrobelny *et al.*, 2008). In addition to defense, a pheromone-like function has been described for HCN-producing compounds in mating behaviors, such as male courtship and female calling and assessment of male fitness (Fig. 1) (Zagrobelny *et al.*, 2018).

Mammalian cells are able to produce HCN from glycine in a reaction catalyzed by peroxidases (Stelmaszynska, 1986; Borowitz *et al.*, 1997; Gunasekar *et al.*, 2000). Compared with plants and bacteria, HCN signaling and regulatory function in mammals are particularly unknown and underestimated because it is almost exclusively viewed as a poison and environmental toxin. Nevertheless, mammals, including humans, have detectable (nM to low µM) plasma HCN levels (Vinnakota *et al.*, 2012), and active white blood cells produce HCN during phagocytosis (Stelmaszynska, 1986). In the brain, synaptic receptors are activated by HCN, which is generated by opiate agonists, indicating that it may act as a neuromodulator (Fig. 1) (Borowitz *et al.*, 1997; Gunasekar *et al.*, 2004; Siegien and Bogatek, 2006). It has been observed recently that very low concentrations of HCN (from nM to 1 µM), rather than having toxicological effects, induce cellular proliferation and bioenergetics via cytochrome *c* oxidase stimulation and increase cell respiration and ATP production in mammalian cell cultures (Pacher, 2021; Randi *et al.*, 2021).

## HCN in plants

HCN is naturally present in relatively high concentrations in cyanogenic plant species such as almonds or cassava (Poulton, 1990; Moller, 2010), forming cyanogenic compounds that, in the case of the latter, can generate a health problem if not properly eliminated, especially in developing countries in times of famine (White *et al.*, 1998; Kashala-Abotnes *et al.*, 2019). In non-cyanogenic plants, HCN is produced principally during the biosynthesis of ethylene (ET) and the antipathogenic molecule camalexin (Peiser *et al.*, 1984; Yip and Yang, 1988; Wang *et al.*, 2002; Glawischnig, 2007; Bottcher *et al.*, 2009). Therefore, HCN is naturally produced in plants and, apart from its toxic action, it has been shown to influence several physiological processes.

Substantial evidence has related the presence of HCN to the acceleration of germination and, indeed, the HCN content in seeds increases just before germination in many cyanogenic and non-cyanogenic plant species (Esashi *et al.*, 1991). It is well known that cyanohydrins present in the smoke of a wildfire induce a germination burst due to the HCN released,
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** HCN functions in organisms of the different kingdoms of life. Despite its toxicity, HCN is present in living organisms, where functions have been described. The different functions in the different life kingdoms are indicated adjacent to the respective arrows. For explanations and references, see the text.</figcaption></figure>

which regenerates the burned landscape (Nelson et al., 2012; Flematti et al., 2013). Treatments with HCN at low concentrations (mM) stimulate seed germination by breaking dormancy in diverse plants, including fruit trees, cereals, sunflower, and Arabidopsis, concomitantly with transient reactive oxygen species (ROS) production and protein carbonylation, and, in some cases, the ET signaling pathway has been shown to be necessary for dormancy break (Gotor et al., 2019, and references therein). An RNA-seq analysis of tomato seeds treated with a low concentration of KCN has shown that HCN acts pleiotropically to break seed dormancy (Yu et al., 2022). Specifically, repression of proteins necessary for seed protein storage that may cause mobilization of stored proteins, as well as induction of glycolytic, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation enzymes, was observed. Therefore, carbon and energy metabolism acceleration is occurring. Finally, hormones such as abscisic acid and gibberellin were also affected in KCN-treated tomato seeds.

Hormones, including ET, are central components of the plant response to pathogens (Zhang et al., 2018). As ET and

HCN biosynthesis are intimately linked, a relationship between HCN and plant immunity is not unlikely and will be discussed below.

### HCN-mediated signaling in plants

Plants possess two HCN-detoxifying enzymatic activities, namely β-cyanoalanine synthases (CASs; EC 4.4.1.9) and sulfurtransferases (STRs; EC 2.8.1.1), which use either cysteine or thiosulfate/mercaptopyruvate to incorporate HCN and convert it into less toxic molecules. The main HCN-detoxifying enzyme in *Arabidopsis thaliana* is CAS, with mitochondrial-CAS-C1 activity accounting for ~70% of the total CAS activity in this plant (Hatzfeld et al., 2000; Machingura et al., 2016; Arenas-Alfonseca et al., 2018b). Although *cas-c1* T-DNA insertion mutants accumulate between 20% and 40% more HCN in their tissues than wild-type plants, their only phenotypic difference is that they present dwarf root hairs that are unable to elongate (Garcia et al., 2010). Moreover, it has been shown that this defect is not dependent on ROS
production or the inhibition of the NADPH oxidase RHD2 that produces the superoxide anion at the tip of the elongating root hair and drives its polar growth (Arenas-Alfonseca et al., 2018a, b). On the other hand, the mutation in *CAS-C1* activates the immune response of plants when infected by bacterial and viral biotrophic pathogens, indicating that endogenously produced HCN might also contribute to regulating the plant immune system (Garcia et al., 2013; see below). Overexpression of *CAS* genes in Arabidopsis has also been shown to be important in salt stress resistance, and alternative oxidase (AOX) seems to be essential in this process (Xu et al., 2023).

HCN can thus act as a signal molecule whose amount is finely regulated in plant tissues and whose action mechanism is important to uncover. It is a small molecule able to penetrate membranes easily due to its lipid solubility. It is enzymatically generated and toxic in excess; therefore, detoxifying activities are necessary to maintain HCN at non-toxic levels. These features are shared by known gasotransmitter signaling molecules, such as ROS, and reactive sulfur and nitrogen species. Their mode of action includes post-translational modifications (PTMs) in proteins at the -SH groups of cysteines,

namely oxidation, persulfidation, and nitrosylation (Aroca et al., 2018, and references therein). PTMs influence the physicochemical properties of the proteins (folding, conformation, subcellular distribution, stability, and activity) and therefore their biological activity. As will be discussed below, HCN *per se* is capable of reacting with cysteine residues in the form of a disulfide bridge to form an organic thiocyanate (Gawron, 1966). We have shown very recently for the first time in any organism that *S*-cyanylation exists naturally in plants (Garcia et al., 2019). The importance of this new PTM is unexplored and represents an exciting challenge. Box 1 summarizes the synthesis, detoxification, and signaling role of HCN in plants, including the *S*-cyanylation as a novel HCN-driven mechanism of action.

Hence, HCN plays a role in different biological processes in plants, which deserves special attention because it has driven a change in the understanding of HCN from a poison to a signaling molecule. These findings are of special relevance because they represent a new mechanism for triggering fast and efficient responses aimed at restoring homeostasis and/or adapting plant physiology to a changing environment,

**Box 1. Synthesis, detoxification, and signaling role of HCN in plants.**

<figure><img src="image_3.png" /></figure>

The intracellular levels of HCN are determined by its production as a co-product in the biosynthesis of ethylene and phytoalexins or in the hydrolysis and degradation of glucosinolates and cyanogenic glycosides, in equilibrium with the mechanism of enzymatic detoxification or its reaction with other biomolecules. The transient or permanent accumulation of HCN induces its reaction with small biomolecules or with proteins and enzymes, altering its functionality and acting as a signaling molecule in the regulation of pathophysiological processes in the plant.
especially important in the current climatic change scenario
and the emergence of new plant diseases.

HCN in plant defense

Cyanogenic compounds constitute an excellent defensive bar-
rier against animal, microbial, and fungal attack, and exist in
>3000 plant species. The core of plant cyanogenesis is based on
the enzymatic hydrolysis of cyanohydrins (α-hydroxynitriles)
to generate carbonyl compounds (aldehyde or ketone) with
free toxic HCN as a consequence of tissue disruption triggered
by herbivores or by physical injury (Morant et al., 2008).

The cyanogenesis process in cyanogenic plants has been es-
tablished by numerous studies, and a canonical pathway has
been proposed: the synthesis of cyanogenic glycosides starts
with the conversion of aromatic (Phe, Tyr) or aliphatic (Val, Ile,
Leu, Trp) amino acids into the respective oximes catalyzed by
cytochromes P450 of the CYP79 family (CYP79D1/D2/A1).
Consecutively, oximes are converted into α-hydroxynitriles
by the action of cytochromes P450 of the CYP71, CYP736,
CYP706, and CYP83 families, to be then glycosylated by
UDP-glycosyltransferases (UGT85B/K). CNglcs are formed
as inactive precursors and need to be enzymatically activated.
After tissue disruption mediated by feeding herbivores, CNglcs
are brought into contact with β-glucosidases that break the
β-glucosidic bond, thereby releasing α-hydroxynitriles (cya-
nohydrins) with defensive properties (Zagrobelny et al., 2004).
Finally, cyanohydrins are hydrolyzed either spontaneously or
by hydroxynitrile lyases (HNLs), dissociating into HCN and
the corresponding aldehyde or ketone (Gleadow and Møller,
2014).

HCN and cyanohydrins are too reactive to be deployed di-
rectly as pre-formed defenses. The storage of inactive precursor
CNglcs permits the generation of appropriate chemically ac-
tive compounds against herbivores in the right tissue and at
the right time: activating enzymes that are compartmentalized
separately to their substrates so that they do not mix until the
plant has been chewed or damaged (Miller and Conn, 1980;
Poulton, 1990; Conn, 2008; Møller, 2010; Gleadow and Møller,
2014). Although CNglcs are the most common cyanogenic
compounds, derivatives from the 4-OH-ICN pathway exclu-
sively found in the Brassicaceae family also produce cyanohy-
drins, which can then be hydrolyzed by the action of the HNL
enzyme, releasing HCN (Rajniak et al., 2015; Pastorczyk et al.,
2020). In the initial step of this pathway, two redundant P450
monooxygenases, CYP79B2 and CYP79B3, convert trypto-
phan into indole-3-acetaldoxime (IAOx), which then acts as
the substrate taken by CYP71A12 to produce indole cyanohy-
drin. At this point, a flavin-dependent oxidoreductase, termed
FOX1, catalyzes the conversion of IAOx to indole-carbonyl-
nitrile (ICN), a substrate of the CYP82C2 enzyme that can
be transformed into 4-OH-ICN. Additionally, 4-OH-ICN is
the base for downstream cyanohydrin metabolite production
(Dixit et al., 2022).

The role of cyanogenic plants in the control of phytopha-
gous arthropod pests has been widely studied (Boter and Diaz,
2023, and references therein). In general, insect damage neg-
atively correlates with cyanogenic and phenolic compounds,
and some studies have even demonstrated that insects such
as Mexican bean beetles (*Epilachna varivestis*) choose plants
with genotypes deficient in cyanide release as a source of
nutrients or as a place to lay their eggs (Balhorn *et al.*, 2006).
Recent studies provide evidence that the benefits of HCN
defense against herbivory on white clover (*Trifolium repens*)
are temperature dependent (Fadoul *et al.*, 2023). In contrast,
there is just one publication validating the alternative cya-
nogenic pathway to expand plant defenses against pests in a
non-cyanogenic plant such as *A. thaliana*. Arnaiz *et al.* (2022)
demonstrated the up-regulation of different enzymes, such as
β-glucosidases, α-HNLs, and FOX1, for the release of HCN in
response to phytopredators such as the spider mite (*Tetranychus
urticae*). Moreover, a reduction in leaf damage determined in
the AtHNL-overexpressing lines reflected the mites' reduced
ability to feed on leaves, which consequently limited mite fe-
cundity. Interestingly, it was recently identified how the in-
teraction of plant-predator co-evolution has led to an arms
race where insects induce β-cyanoalanine synthase for HCN
detoxification as protection against the defenses of Arabidopsis
plants (Dixit *et al.*, 2022; Nguyen, 2022).

HCN has also been proposed as a signaling molecule that
could act indirectly in the plant response to pathogenic bacteria,
viruses, or fungi. PR-1 and several salicylic acid (SA)-regulated
genes are transcriptionally induced in the basal state in cas-c1
mutant plants (Garcia et al., 2013, 2014). It has been extensively
demonstrated that cas-c1 mutant plants are more resistant than
wild-type plants to biotrophic and hemibiotrophic pathogens
such as the bacterium Pseudomonas syringae and beet curly top
virus (BCTV) (Garcia et al., 2013, 2014). Furthermore, double
mutants atrbohD3 cas-c1 show a notably lower susceptibility to
P. syringae pv. tomato DC3000 infection than the simple mu-
tant atrbohD3, suggesting that the accumulation of HCN due
to the cas-c1 mutation is capable of activating plant defenses
in a similar way to the effect of ROS produced by NADPH
oxidase (Arenas-Alfonseca et al., 2021). Interestingly, the cas-
c1 mutant does not produce more O2- than wild-type plants;
therefore, the bypass of NADPH oxidase does not depend on
ROS production in the presence of HCN (Arenas-Alfonseca
et al., 2021). Proteomic analyses of wheat and barley varieties
with different sensitivities to the fungus Fusarium graminearum
reveal a correlation between β-CAS activity and fungal resist-
ance of the different lines examined (Geddes et al., 2008; Zhang
et al., 2013). Moreover, exogenously applied HCN has been
shown to enhance rice defense against the fungus Magnaporthe
grisea, whose production is triggered by a hypersensitive reac-
tion (HR) (Iwai et al., 2006; Seo et al., 2011). The underlying
mechanism of cyanide production in plant immunity against
these kinds of pathogens is very complex and requires further
study.
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** HCN and plant defense. (A) Exogenous treatments with HCN or the production of HCN by the plant-associated cyanogenic microbiome in the rhizosphere activate plant defense against a wide range of pathogens. (B) Plant endogenously produced HCN and HCN-producing molecules have been shown to promote plant defense against herbivores and biotrophic pathogens.</figcaption></figure>

In addition to its action in defense, cyanide also plays an important role during compatible and incompatible plant-bacteria interactions. Endogenous HCN content is oppositely regulated in virulent or avirulent interactions with the hemi-biotrophic bacterium *P. syringae*. During an incompatible interaction between Arabidopsis plants and the avirulent *P. syringae* pv. tomato DC3000 avrRpm1 (Pst DC3000 avrRpm1), there is an early decrease in the expression of *CAS-C1* that leads to a transient increase in the concentration of HCN, which, analogous to the oxidative burst occurring at early stages of the infections, can be considered an HCN burst (Garcia *et al.*, 2013, 2014, 2019).

As mentioned above, a wide range of organisms, such as fungi, bacteria, lichens, millipedes, arthropods, and insects, are also cyanogenic (Zagrobelny *et al.*, 2008). In this context, HCN is also implicated in the plant-associated microbiome in the rhizosphere. Cyanogenic rhizobacteria produce HCN, which can also be used as an effective herbicide to inhibit the growth of weed seedlings in *Lactuca sativa* (Kremer and Souissi, 2001), an alternative to the toxic herbicides in current use. On the other hand, strong growth inhibition has been observed in various pathogenic fungi, weeds, insects, termites, and nematodes by the effect of cyanogenic bacteria (Sehrawat *et al.*, 2022). Numerous *Trichoderma* species show antagonism against phytopathogenic microorganisms producing HCN, siderophores, antibiotics, and fungal cell wall-lysing enzymes such as cellulases, ligninases, chitinases, and proteases (Jamil, 2021; Saeed *et al.*, 2021). HCN produced by *Trichoderma* acts as a defense regulator and inhibitor against different phytopathogens, including *Aspergillus niger*, *A. flavus*, *Fusarium oxysporum*, and *Alternaria alternata* (Blumer and Haas, 2000; Cucu *et al.*, 2019; Zain *et al.*, 2019; Jamil, 2021).

In fact, other signaling molecules, such as NO, which has chemical characteristics very similar to HCN, have been suggested to be further involved in the induced systemic resistance of plants. Pescador *et al.* (2022) reported that NO of *Trichoderma* fungi causes induced systemic resistance in Arabidopsis plants and is effective against a wide range of pathogens. Therefore, the use of HCN produced by bacteria and fungi as a biocide may be a pathway for its use in sustainable agriculture (Rijavec and Lapanje, 2016; Sendi *et al.*, 2020; Sehrawat *et al.*, 2022). Figure 2 represents a scheme of the implication of HCN in plant defense.

## Mechanisms of action of HCN

The measured HCN content in plant tissues ranged from 25–150 ppb in Arabidopsis leaves and roots to 25–1000 ppm in cyanogenic plants such as cassava and bamboo (Haque and Bradbury, 2002). In cyanogenic plants, the release of cyanide depends on the action of hydrolytic enzymes (β-glucosidases) to break down the cyanoglycoside (Cressey and Reeve, 2019).

The function of HCN as a signaling molecule, in a similar way to other signaling molecules such as H₂S, must lie in the transient accumulation of cyanide and its chemical reactivity with other biomolecules. At least three mechanisms of action can be suggested to explain the signaling role of HCN in eukaryote cells (Fig. 3): (i) reaction with other small biomolecules; (ii) binding with metal centers and metalloproteins; and (iii) modification of protein cysteines to form the corresponding thiocyanate (Prot-S-C≡N).
A Nucleophilic Cyanide Transfer

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /></figure>

or

<figure><img src="image_7.png" /></figure>

+

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

Ketone

Aldehyde

Cyanohydrin

C

<figure><img src="image_11.png" /></figure>

+

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

Cysteine

<figure><img src="image_15.png" /></figure>

S-cyano-L-cysteine

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

2-iminothiazolidine-4-carboxylic acid
(ITCA)

2-aminothiazoline-4-carboxylic acid
(ATCA)

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

GSH

+

<figure><img src="image_22.png" /></figure>

GS-CN

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

+

<figure><img src="image_25.png" /></figure>

Glutamic acid

(2-amino-4,5-dihydrothiazole-4-carbonyl)glycine

<caption>**Fig. 3.** Cyanide reactivity with small molecules. (A) Schematic reaction of cyanide with electrophilic molecules. (B) Cyanohydrin formation by reaction with ketone and aldehyde molecules. (C) Cyanide reaction with disulfide bridges of cystine and oxidized glutathione.</caption>
HCN is weakly acidic, with a pKa of 9.2, and thus ionizes in
aqueous solution to give the cyanide anion N≡C:−. The lone
pair of electrons and the negative charge in the carbon atom
make the cyanide anion a strong nucleophile that easily reacts
with molecules by nucleophilic substitution reactions (Fig. 3A);
thus, cyanide easily reacts with carbonyl compounds, especially
aldehydes and ketones, to form cyanohydrins or hydroxyni-
triles (Fig. 3B). Cyanogenic plants contain the hydroxynitrile
lyase enzyme that catalyzes the opposite reaction and breaks
down cyanohydrin to release cyanide and aldehyde or ketone,
and this reaction is a self-defense mechanism to protect plants
containing cyanogenic glycosides from microbial and insect at-
tack (Zagrobelny et al., 2008). Although mainly described in
cyanogenic plants, HNL activity has also been described in
Arabidopsis to act in response to spider mite infestation (Arnaiz
et al., 2022). Cyanide can also react with cystine and mixed
disulfides in general by nucleophilic attack on sulfur to yield a
thiol and a thiocyanate, which further react to form more stable
thiazoline heterocyclic compounds; thus, cystine reacts with
cyanide to form cysteine and 2-aminothiazoline-4-carboxylic
acid (ATCA) or its tautomer 2-iminothiazoline-4-carboxylic
acid (ITCA) (Fig. 3C) (Boyde, 1968). These thiazolines can be
detected in urine samples from humans with high dietary in-
take of cyanide and are used in forensic science (Lundquist
et al., 1995; Nishio et al., 2022). Similarly, the oxidized form of
glutathione (GSH), the most abundant thiol in eukaryote cells,
can also react in the same way with cyanide and form reduced
GSH and cyano-glutathione (GS-CN), which can cycle be-
tween the N-terminus of the cysteine residue and the CN
bond, which cleaves the γ-glutamyl-cysteine peptide bond to
produce the corresponding 2-aminothiazolidine (ATOEA) (or
the tautomer 2-iminothiazolidine) and glutamic acid (Degani
and Patchornik, 1974). The analysis of ATOEA in plasma has
also been used as a biomarker of cyanide exposure (Gyamfi
et al., 2019). The presence of ATCA or ATOEA in plants has
not been analyzed thus far, but the abundance of GSH in plant
cells makes it plausible that it may act as a cyanide-detoxifying
molecule or, vice versa, that cyanide accumulation during ET
burst may act by reducing GSH levels under stress conditions.
Cyanide can also react with reduced sulfur species such as
polysulfides and, in the presence of an oxidizing agent, it can
react with certain oxidized forms of sulfur such as sulfite and
thiosulfate to produce thiocyanate (Bartlett and Davis, 1958).

The cyanide anion reacts with transition metals to form
M-CN bonds and generate complexes with coordination num-
bers from two to eight (Hanusa, 2005). Cyanide complexes are
commonly formed by transition metals with oxidation states
(+3) and (+2), including iron, copper, mercury, zinc, nickel,
cadmium, cobalt, other transition group metals, and, of course,
gold and silver. The capacity to form very stable metal com-
plexes is the basis of cyanide toxicity since it can bind to heme
a₃ of cytochrome c oxidase, inhibiting the utilization of mo-
lecular oxygen (Keilin et al., 1939); thus, cyanide affects com-
plex IV and inhibits mitochondrial electron transport and ATP

generation (Keilin et al., 1939). Although cyanide can inhibit
cytochrome c oxidase activity, it has been recently reported that
at a low concentration, cyanide stimulates mitochondrial elec-
tron transport and ATP generation associated with the removal
of inhibitory glutathionylation post-translational modifica-
tion of the 30 kDa and 57 kDa subunits (Randi et al., 2021).
Cyanide is also able to interact with other heme proteins, such
as hemoglobin and myeloperoxidase (MPO), which catalyze
the oxidation of chloride, bromide, and thiocyanate to pro-
duce strongly oxidizing molecules such as hypochlorous acid
(HOCl), hypobromous acid (HOBr), or hypothiocyanous acid
(HOSCN) during the neutrophil respiratory burst (Rossifanelli
et al., 1964; Winterbourn et al., 2016). Interestingly, MPO is one
of the mammalian enzymes described that produces cyanide
from glycine (Zgliczynski and Stelmaszynska, 1979). Nitrite
and sulfite reductases are key nitrogen and sulfur assimilatory
enzymes found in bacteria, fungi, and plants; they contain an
Fe–heme siroheme linked to the [4Fe-4S] cluster, and their
interaction with cyanide positively shifts the redox potential
of the siroheme, altering the redox capability of the enzymes
(Liu et al., 2014). Other metalloproteins are also susceptible
to interaction with cyanide in vitro, such as Fe-superoxide re-
ductase (FeSOD), Cu,Zn-superoxide dismutase (Cu,ZnSOD),
and Mn-superoxide dismutase (Clarkson et al., 1989; Iqbal
and Whitney, 1991; Shearer et al., 2003). Plant photosynthesis
is also very sensitive to cyanide, which is a potent inhibitor
of electron transfer to PSI since it can bind Cu-plastocyanin
(Berg and Krogmann, 1975).

The high affinity of cyanide for cobalamin or vitamin B12,
a coordination complex of cobalt ions in the center of a cor-
rin heterocyclic ring, makes this molecule a more effective and
common antidote in cyanide poisoning (Borron et al., 2006).
In plant tissue, treatment with hydroxocobalamin can reverse
the inhibition of root hair development due to cyanide accu-
mulation in mutant lines defective in the cyanide detoxifica-
tion enzyme β-cyanoalanine synthase (Garcia et al., 2010).

The recognized gaseous signaling molecules carbon mon-
oxide (CO), NO, and H2S exert their cellular effects by inter-
acting with cellular and molecular targets. The three of them
can react with transition metals to give metal complexes, but,
in addition to other mechanisms, NO and H2S carry out their
function through the post-translational modification of cys-
teine thiol residues in protein targets, such as S-nitrosylation
and persulfidation, respectively. HCN may react with protein
disulfide by nucleophilic displacement and form S-cyanylated
cysteine residues. This modification can be detected in human
plasma proteins, such as immunoglobulin G and serum al-
bumin, after cyanide poisoning or in individuals who smoke
(Fasco et al., 2007; Grigoryan et al., 2016). The presence of
S-cyanylation in a wide variety of proteins was described in
Arabidopsis wild-type plants as well as in a mutant line defec-
tive in the β-cyanoalanine synthase enzyme, which accumu-
lated higher levels of cyanide (Garcia et al., 2010). Using two
different technical approaches, a set of 163 proteins were shown
<figure><img src="image_26.png" /><figcaption>**Fig. 4.** Conclusions and upcoming.</figcaption></figure>

to be susceptible to *S*-cyanylation. The first approach took ad-
vantage of the described characteristic of *in vitro* *S*-cyanylated
proteins that can be cleaved in two parts under alkaline condi-
tions, the amino acid backbone from the N-terminus of the
cyanylated cysteine residue on one side and the cycled imino-
thiazolidine-derived peptide with the C-terminus of the pro-
tein on the other (Catsimpoolas and Wood, 1966; Fasco *et al.*,
2007). Comparative analysis of the protein profiles in wild-type
and *cas-c1* lines revealed 88 cyanylated proteins involved in im-
portant metabolic pathways, such as glycolysis and the TCA and
Calvin cycles. Enolase 1 and 2, part of the phosphoryruvate
hydratase complex, showed a higher (>100-fold) cyanylation
change in *cas-c1* compared with the wild type, with Cys346
being the modified residue (Garcia *et al.*, 2019). Alternative
approaches used LC-MS/MS to analyze protein extracts from
enriched mitochondrial preparations from root tissues and iden-
tified an additional 75 *S*-cyanylated proteins in roots. To date, a
total of 163 *S*-cyanylated proteins have been described in plants
(Garcia *et al.*, 2019). Some of the more interesting findings were
that most of the enzymes of the *S*-adenosylmethionine cycle,
including Met synthase 1 and 2 (MS1 and MS2) and DNA
methyltransferase 2 (DMT2), were modified. This may have im-
portant signaling and regulatory aspects related to protein and
DNA methylation. Indeed, MS1 and DNA methylation are re-
lated to immune priming (González and Vera, 2019), which is
a status that can deploy defense mechanisms more rapidly and
robustly following a pathogen infection, with a minimal cost to
the physiology of plants because the defensive response is initi-
ated only if necessary. This may result in noteworthy implica-
tions that require further attention.

**Conclusion**

Information gained using model plants has changed the per-
ception of HCN from a poison to a useful molecule that is able

to generate a signal that transduces external (biotic or abiotic
stress) or internal (development or other natural processes) sig-
nals in a response aiming to restore plant (or any organism)
homeostasis and adapt to the new situation. Apart from ag-
riculture (deepening the knowledge of the immune response
and developmental processes in plants regulated by cyanide),
generated tools and knowledge can be useful for exploring
other areas, such as biomedicine (identifying new human path-
ophysiological processes regulated by HCN), microbiology
(identifying pathophysiological processes in bacteria and fungi
regulated by HCN), and biotechnology (manipulating biolog-
ical processes by protein activity alteration by *S*-cyanylation)
(Fig. 4).

**Acknowledgements**

We acknowledge the European Regional Development Fund, Ministerio de Economía y Competitividad, the Agencia Estatal de Investigación and CSIC, and the Junta de Andalucía for funding.

**Conflict of interest**

The authors have no conflict of interest to declare.

**Funding**

This work has been supported by the ERDF “A way of making Europe” and MCIN/AEI/10.13039/ 501100011033 (grants No. BIO2016-76633-P and PID2021-127450NB-I00), CSIC (grant. No. 201840I085) and Junta de Andalucía (grant No. P20_00030).

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