title: 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 (50-300μM). The activity of immunocaptured mitochondrial ATP synthase enzyme isolated from HepG2 and HEK293 cells was stimulated by NaHS at low concentrations (10-100nM). 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.
authors.affiliation: Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, ON, Canada; Department of Surgery, University of Texas Medical Branch, Galveston, TX, USA; Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Katalin
authors.initials: K
authors.lastname: Módis
authors.affiliation: Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, ON, Canada.
authors.forename: YoungJun
authors.initials: Y
authors.lastname: Ju
authors.affiliation: Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Akbar
authors.initials: A
authors.lastname: Ahmad
authors.affiliation: Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, ON, Canada; Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Ashley A
authors.initials: AA
authors.lastname: Untereiner
authors.affiliation: Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, ON, Canada.
authors.forename: Zaid
authors.initials: Z
authors.lastname: Altaany
authors.affiliation: School of Human Kinesiology, Laurentian University, Sudbury, ON, Canada.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA. Electronic address: szabocsaba@aol.com.
authors.forename: Csaba
authors.initials: C
authors.lastname: Szabo
authors.affiliation: Cardiovascular and Metabolic Research Unit, Lakehead University, Thunder Bay, ON, Canada; Department of Biology, Laurentian University, ON, Canada. Electronic address: rui.wang@laurentian.ca.
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D003542:Cystathionine gamma-Lyase / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004734:Energy Metabolism / Q000502:physiology*; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D006862:Hydrogen Sulfide / Q000378:metabolism*; D008099:Liver / Q000378:metabolism / Q000502:physiology; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D011499:Protein Processing, Post-Translational / Q000502:physiology
chemical_list: D000255:Adenosine Triphosphate; D025261:Mitochondrial Proton-Translocating ATPases; D003542:Cystathionine gamma-Lyase; D003545:Cysteine; D006862:Hydrogen Sulfide
keywords: ATP synthase; Bioenergetics; Burn; Burn injury; Cysteine; H(2)S; Hydrogen sulfide; Mitochondria; S-Sulfhydration