S- Sulfhydration of ATP synthase by hydrogen sulfide stimulates mitochondrial bioenergetics

Abstract

Mammalian cells can utilize hydrogen sulfide (H2S) to support mitochondrial respiration. The aim of our study was to explore the potential role of S-sulfhydration (a H2S-induced posttranslational modification, also known as S-persulfidation) of the mitochondrial inner membrane protein ATP synthase (F1F0 ATP synthase/Complex V) in the regulation of mitochondrial bioenergetics. Using a biotin switch assay, we have detected S-sulfhydration of the α subunit (ATP5A1) of ATP synthase in response to exposure to H2S in vitro. The H2S generator compound NaHS induced S-sulfhydration of ATP5A1 in HepG2 and HEK293 cell lysates in a concentration-dependent manner (50–300 μM). The activity of immunocaptured mitochondrial ATP synthase enzyme isolated from HepG2 and HEK293 cells was stimulated by NaHS at low concentrations (10–100 nM). Site-directed mutagenesis of ATP5A1 in HEK293 cells demonstrated that cysteine residues at positions 244 and 294 are subject to S-sulfhydration. The double mutant ATP synthase protein (C244S/C294S) showed a significantly reduced enzyme activity compared to control and the single-cysteine-mutated recombinant proteins (C244S or C294S). To determine whether endogenous H2S plays a role in the basal S-sulfhydration of ATP synthase in vivo, we compared liver tissues harvested from wild-type mice and mice deficient in cystathionine-gamma-lyase (CSE, one of the three principal mammalian H2S-producing enzymes). Significantly reduced S-sulfhydration of ATP5A1 was observed in liver homogenates of CSE−/− mice, compared to wild-type mice, suggesting a physiological role for CSE-derived endogenous H2S production in the S-sulfhydration of ATP synthase. Various forms of critical illness (including burn injury) upregulate H2S-producing enzymes and stimulate H2S biosynthesis. In liver tissues collected from mice subjected to burn injury, we detected an increased S-sulfhydration of ATP5A1 at the early time points post-burn. At later time points (when systemic H2S levels decrease) S-sulfhydration of ATP5A1 decreased as well. In conclusion, H2S induces S-sulfhydration of ATP5A1 at C244 and C294. This post-translational modification may be a physiological mechanism to maintain ATP synthase in a physiologically activated state, thereby supporting mitochondrial bioenergetics. The sulfhydration of ATP synthase may be a dynamic process, which may be regulated by endogenous H2S levels under various pathophysiological conditions.

Upregulation of Cystathionine-β-Synthase in Colonic Epithelia Reprograms Metabolism and Promotes Carcinogenesis

“…This is likely due to the increased CBS-dependent synthesis of H 2 S by CBS2 cells (Fig 2D, E) and the accumulation of lanthionine (Table 1). H 2 S can directly stimulate cellular bioenergetics by contributing electrons to SQR in complex II to reduce coenzyme 10 to ubiquinol and, indirectly, by the post-translational persulfidation of F1F0 of ATP synthase/Complex V with the result of enhancing ATP production (37–39). Persulfidation, also known as sulfhydration is the modification of cysteine residues on the target protein by H 2 S to form a persulfide or–SSH group.…”

H 2 S-induced S -sulfhydration of lactate dehydrogenase a (LDHA) stimulates cellular bioenergetics in HCT116 colon cancer cells

“…The assay was conducted as described elsewhere [16] with modifications. Briefly, human recombinant LDHA (1 μg; Sigma) was suspended in HEN buffer [250 mM Hepes (pH 7.7), 1 mM EDTA, and 0.1 mM neocuproine] supplemented with 100 μM deferoxamine and centrifuged at 14,000 rpm for 20 min at 4°C.…”

“…The functional consequence of this response is stimulation of cellular bioenergetics, tumor growth and proliferation (overviewed in [7]). The mechanisms involved in the stimulation of mitochondrial function by H 2 S are multiple; they involve direct electron donation to the mitochondrial electron transport chain [8–10], inhibition of mitochondrial cAMP phosphodiesterases, followed by cAMP-stimulated increases in mitochondrial electron transport [11], mitochondrial antioxidant effects [12–15], stimulation of mitochondrial DNA repair [6,15] as well as a direct stimulation of mitochondrial ATP synthase via post-translational modification ( S -sulfhydration) [16]. All of these effects occur at low-to-intermediate concentrations of H 2 S, while at higher concentrations, inhibitory effects of H 2 S on mitochondrial function become apparent - primarily mediated by the inhibition of mitochondrial Complex IV (cytochrome c oxidase) by H 2 S [17–19].…”

Protein S‐sulfhydration by hydrogen sulfide in cardiovascular system

“…And there was lower S‐sulfhydration and weaker activity of ATP5A1 in CSE −/− mice. This suggests that S‐sulfhydration of ATP5A1 might be beneficial for the maintenance of ATP synthase homeostasis in mitochondrial energy disposal as well as antioxidant activity and redox signalling (Módis et al, ). Most of the findings revealed that S‐sulfhydration was the main post‐translational modification induced by H 2 S and played an important role in ROS production and redox signalling in the cardiovascular system.…”