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Matsunaga et al., unpublished observation), and signal transduction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and enzymatic activity regulation (Kasamatsu et al., unpublished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.