Supersulfide and NRF2
Recently, ”supersulfides” have been recognized as a new entity of biomolecules (Zhang et al., 2023). Supersulfides are defined as molecules possessing catenated sulfur, and they are present in the form of low-molecular-weight metabolites and in the cysteine residue side chains of proteins. Typical examples are cysteine persulfide (CysSSH) and glutathione persulfide (GSSH) as reduced forms, and cystine trisulfide (CysSSSCys) and glutathione trisulfide (GSSSG) as oxidized forms. A unique chemical property of supersulfides is dual redox reactivity to both electrophiles and nucleophiles, which enables supersulfides to get involved in various biochemical reactions. Because pKa value of the hydropersulfide moiety (-SSH) is lower than that of simple thiol moiety (hydrosulfide moiety; -SH), CysSSH and GSSH are more reactive to electrophiles such as oxidative stress than cysteine (CysSH) and glutathione (GSH) (Ida et al., 2014). Physiological roles of supersulfides include antioxidant functions (Ida et al., 2014; Millikin et al., 2016), anti-inflammatory functions (Zhang et al., 2019; Matsunaga et al., unpublished observation), and signal transduction (Nishida et al., 2012; Nishimura et al., 2019). Supersulfides also contribute to energy metabolism (Akaike et al., 2017; Marutani et al., 2021; Alam et al., 2023), protein quality control (Dóka et al., 2020) and enzymatic activity regulation (Kasamatsu et al., unpublished observation).
Several enzymes have been identified to synthesize supersulfides. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) catalyze transsulfuration, which acts as a conversion pathway of methionine into cysteine, and also reportedly produce hydrogen sulfide/hydropersulfide (H2S/HS). In addition to these activities, CBS and CSE have been shown to possess activities to generate CysSSH from cystine (Ida et al., 2014), which is a very unique reaction because a disulfide bond in cystine is converted to HS via C-S lyase-like reaction without consuming reducing equivalent (Figure 5). 3-Mercaptopyruvate sulfertransferase (3-MST) was also reported as the third enzyme generating H2S and supersulfides (Kimura et al., 2017). However, simultaneous disruption of the three enzymes, CBS, CSE and 3-MST, in mice does not eliminate supersulfide production in vivo (Zainol Abidin et al., 2023), strongly suggesting the presence of alternative compensatory mechanisms for supersulfide production. Indeed, cysteinyltRNA synthetase (CARS) has been found to possess cysteine persulfide synthesizing activity as a moonlighting function (Akaike et al., 2017). CARS1 and CARS2 are cytoplasmic and mitochondrial isoforms, respectively, and both isoforms possess four motifs well-conserved among species: two of them are for zinc coordination and the other two are for binding of pyridoxal phosphate (PLP). The former are essential for the cysteinyl-tRNA synthesizing activity and thereby related to protein translation, while the latter are essential for cysteine persulfide synthesizing activity. CARS1 and CARS2 are considered to generate low-molecular-weight supersulfides as well as to conjugate cysteine persulfide with tRNA to generate persulfidated cysteinyl-tRNA, allowing cysteine persulfide to be incorporated into a nascent polypeptide in the ribosome. Although the functional significance of the protein supersulfidation at the translation stage remains to be elucidated, supersulfide production in mitochondria by CARS2 has been shown essential for the mitochondrial function.
Impairment of CARS2-mediated supersulfide production depolarizes mitochondrial membrane potential and reduces oxygen consumption (Akaike et al., 2017; Alam et al., 2023), suggesting an essential role of supersulfides in the mitochondrial energy metabolism. Although a recombinant CARS protein synthesizes CysSSH, accumulation of H2S/HS, rather than CysSSH, was observed in cells. However, when mitochondria are partly depleted in the cells by ethidium bromide treatment, H2S/HS was decreased, but instead CysSSH was increased (Akaike et al., 2017). These results unequivocally indicate that CysSSH is reduced to H2S/HS in the ETC function-dependent manner, implying that supersulfides generated in mitochondria serve as electron acceptors (Figure 5). The consequently-generated H2S/HS is oxidized to supersulfides by sulfide:quionone oxidoreductase (SQOR), which is considered to prevent accumulation of H2S/HS and avoid mitochondrial inhibition by sulfide toxicity (Marutani et al., 2021). Sulfur oxidation enzymes residing in mitochondria, ETHE1 and SUOX, also oxidize supersulfides to generate thiosulfate (HS2O3), sulfite (HSO3) and sulfate (HSO4) using molecular oxygen (Figure 5) (Luna-Sánchez et al., 2017; Ziosi et al., 2017). If the supersulfide synthesis in mitochondria is impaired, mitochondrial electrons that should be accepted by supersulfides are expected to be transferred to oxygen, leading to the generation of ROS. Thanks to the presence of supersulfides, electrons leaked from the ETC are not accepted by oxygen but by supersulfides and return to the ETC via SQOR. The supersulfide-mediated electron flow is considered as a rescue circuit for leaked electrons, that is, a mechanism avoiding excessive generation of ROS and ensuring the efficiency of the ETC. Therefore, the mitochondrial supersulfide production and the subsequent sulfur oxidation pathway play a critical role in the mitochondrial energy metabolism.
Consistent with the observation that sulfur metabolism makes a substantial contribution to the mitochondrial respiration, cysteine supply is critical for the mitochondrial activity (Alam et al., 2023). One of the supply routes of cysteine is to uptake extracellular cystine via a cystine transporter xCT. Another route is cysteine intracellularly-synthesized from methionine via transsulfuration pathway. As mentioned above, NRF2 directly activates Slc7a11gene, which encodes xCT (Sasaki et al., 2002), and thereby increases cellular pool of cysteine, ultimately resulting in the increased production of supersulfides. Importantly, NRF2-mediated mitochondrial activation is canceled either by inhibition of xCT, suppression of CARS2-mediated supersulfide production, or inhibition of the mitochondrial sulfur oxidation pathway, supporting the idea that NRF2 activates mitochondria though promoting the mitochondrial sulfur metabolism (Figure 5). From a different point of view, the role of NRF2 in the mitochondria can be interpreted as another mode of antioxidant function of NRF2: avoiding excessive production of ROS and protecting cells from the oxidative stress derived from mitochondria during oxygen respiration.