Sulfide:quinone oxidoreductase drives mitochondrial supersulfide metabolism to regulate bioenergetics and longevity in eukaryotes

This study demonstrates that the mitochondrial enzyme sulfide:quinone oxidoreductase (SQR) is essential for maintaining sulfur respiration, bioenergetics, and longevity across eukaryotes by oxidizing sulfide into supersulfides, as evidenced by impaired mitochondrial function and reduced lifespan in SQR-deficient yeast and mice models that could not be rescued by supersulfide supplementation.

The Gist

The Big Picture: The "Sulfur Power Plant"

Imagine your body's cells are like bustling cities. Inside every city, there are tiny power plants called mitochondria. Their main job is to burn fuel (like sugar) to create electricity (energy/ATP) that keeps the city running.

For a long time, scientists thought these power plants only ran on "sugar fuel." But this new research discovers a secret backup generator that runs on sulfur.

The star of this story is a tiny machine inside the power plant called SQR (Sulfide:quinone oxidoreductase). Think of SQR as a specialized translator or a conductor that knows how to turn sulfur into electricity.

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The Story in Three Acts

Act 1: The Broken Translator (The Yeast Experiment)

First, the scientists looked at a tiny organism called yeast (a single-celled fungus). They created a version of yeast where they removed the SQR machine.

• The Sugar Test: When they fed the yeast sugar, everything was fine. The yeast grew normally. This is like a city that can still run on its main power grid.
• The Glycerol Test: When they switched the fuel to glycerol (which requires the mitochondria to work hard), the yeast without SQR crashed. They couldn't grow.
• The Poison Test: The yeast without SQR also couldn't handle sulfur-based poisons (like hydrogen sulfide). They got sick and died quickly.
• The Lifespan: Most importantly, the yeast without SQR didn't just get sick; they aged faster. They died young.

The Analogy: Imagine a car that runs perfectly on regular gas (sugar) but has a broken turbocharger (SQR). If you try to drive it up a steep mountain (glycerol metabolism), the engine stalls. Also, without that turbo, the car can't handle the special "nitro" fuel (sulfur) that could actually make it go faster and last longer.

Act 2: The Mouse Model (The "Lost" Translator)

Next, the scientists wanted to see if this happened in complex animals like mice. They created a special mouse where the SQR machine existed, but it was lost. It was stuck in the wrong part of the cell (the cytoplasm) and couldn't get into the power plant (mitochondria).

• The Result: These mice were emaciated (very thin) and died young after they were weaned from their mothers.
• The Chemical Clue: Inside these mice, they found a massive buildup of sulfur compounds (like hydrogen sulfide and persulfides). It was like a factory where the waste disposal truck (SQR) broke down, so toxic waste piled up everywhere.

The Analogy: It's like a city where the garbage trucks (SQR) are stuck in the suburbs and never reach the city center. The city gets flooded with trash (sulfur waste), the power plants can't run efficiently, and the city starts to collapse.

Act 3: The Magic Fix (Supersulfides)

Here is the most exciting part. The scientists found that if they gave supersulfides (a specific type of sulfur molecule) to the healthy yeast, the yeast lived longer.

• The Catch: If they gave the supersulfides to the yeast without the SQR machine, it did nothing. The yeast didn't live longer.
• The Conclusion: The sulfur itself isn't the magic pill. The magic is the combination of the sulfur fuel + the SQR machine. SQR takes the sulfur, processes it, and feeds it directly into the power plant's electron chain to create extra energy.

The Analogy: Think of SQR as a chef and sulfur as a special ingredient.

• If you have the ingredient but no chef (SQR-deficient), you just have a pile of raw food. It doesn't help you.
• If you have the chef but no ingredient, they can't cook.
• But if you have both, the chef cooks a delicious meal that gives you super energy and makes you live longer.

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Why Does This Matter?

1. A New Way to Make Energy: We used to think mitochondria only burned sugar and fat. This paper proves they can also burn sulfur to make electricity. It's like discovering your car can run on electricity and a secret fuel we didn't know about.
2. Aging and Longevity: The study shows that this sulfur-burning process is crucial for living a long, healthy life. Without it, cells get tired and die young.
3. Future Medicine: This could be a game-changer for people with mitochondrial diseases (where the power plant is broken).
   • If a patient's "Complex I" (a part of the power plant) is broken, they usually can't make energy.
   • But this research suggests that if we can boost the SQR pathway (the sulfur route), we might be able to bypass the broken part and generate energy anyway. It's like building a detour road around a collapsed bridge.

The Bottom Line

SQR is the essential conductor that turns sulfur into life-sustaining energy. Without it, our cellular power plants lose efficiency, we age faster, and we can't handle sulfur-based fuels. With it, we unlock a hidden energy source that could help us live longer and treat diseases.

Technical Summary

1. Problem Statement

While Sulfide:quinone oxidoreductase (SQR) is known to oxidize hydrogen sulfide ($H_2S$) and transfer electrons to the mitochondrial electron transport chain (ETC), its precise physiological role in endogenous sulfur metabolism, mitochondrial bioenergetics, and organismal longevity remains poorly understood. Previous studies largely relied on exogenous sulfide donors and viewed SQR primarily as a detoxification enzyme. There is a critical gap in understanding how SQR-mediated oxidation of sulfur species (specifically supersulfides like hydropersulfides) contributes to mitochondrial membrane potential, ATP production, and aging across eukaryotes.

2. Methodology

The study employed a multi-species approach combining genetic models, metabolomics, and bioenergetic assays:

• Yeast Model ($Schizosaccharomyces pombe$):

  • Generated an SQR-deficient strain ($\Delta hmt2$) via homologous recombination.
  • Validated the model using sensitivity assays to Cadmium ($CdCl_2$) and sulfide donors ($NaHS$).
  • Assessed growth on glucose (glycolysis-dependent) vs. glycerol (respiration-dependent) media.
  • Measured mitochondrial membrane potential ($\Delta\Psi_m$) using JC-1 staining and Flow Cytometry (FACS).
  • Conducted chronological lifespan assays (colony-forming units over time).
  • Performed metabolomics (LC-ESI-MS/MS) to quantify sulfur species (cysteine, persulfides, thiosulfate, etc.).
  • Tested lifespan extension via supplementation with $NaHS$ (sulfide donor) and $Na_2S_2$ (hydropersulfide donor).

• Mammalian Model (Mouse):

  • Utilized a mitochondria-selective SQR-deficient mouse model ($Sqrdl^{\Delta N/\Delta N}$) where the mitochondrial targeting sequence was deleted via CRISPR-Cas9, preventing SQR localization to mitochondria while allowing cytoplasmic expression.
  • Generated immortalized Mouse Embryonic Fibroblasts (iMEFs) from these mice.
  • Isolated mitochondria from iMEFs and mouse livers.
  • Measured $\Delta\Psi_m$ using JC-1 (cells) and TMRE (isolated mitochondria) via confocal microscopy.
  • Assessed Oxygen Consumption Rate (OCR) using the XF96 Extracellular Flux Analyzer and Clark-type electrodes.
  • Used specific ETC inhibitors (Rotenone, Atpenin A5, Antimycin A, CATR) and $H_2S$ scavengers (SS19) to dissect the mechanism.

3. Key Contributions

• Discovery of a "Sulfur Respiration" Pathway: The paper establishes that SQR is not merely a detoxifier but a central driver of a sulfur-based energy metabolism pathway where supersulfides (hydropersulfides and polysulfides) serve as electron donors to the ETC.
• Substrate Specificity Revision: The study revises the understanding of SQR substrates, demonstrating that it oxidizes not only $H_2S$ but also hydropersulfides (e.g., CysSSH, GSSH) and polysulfides, which appear to be more efficient electron donors than free $H_2S$.
• Mechanism of Membrane Potential Generation: It elucidates that SQR feeds electrons into the ETC at the level of Ubiquinone (CoQ), downstream of Complex I and II but upstream of Complex III, thereby generating a proton gradient independent of Complex I.
• Evolutionary Conservation: The findings demonstrate that this sulfur-based bioenergetic mechanism is conserved from fission yeast to mammals and is critical for longevity.

4. Key Results

A. Yeast ($\Delta hmt2$) Findings:

• Metabolic Defect: $\Delta hmt2$ yeast grew normally on glucose but failed to grow on glycerol, indicating a specific defect in mitochondrial respiration.
• Bioenergetics: SQR-deficient yeast exhibited reduced mitochondrial membrane potential, lower ATP levels, and a significantly shortened chronological lifespan (surviving <12 days vs. 18 days for WT).
• Metabolomics: $\Delta hmt2$ showed elevated $H_2S$ and massive accumulation of cysteine hydropersulfide (CysSSH) and thiosulfate, confirming SQR's role in oxidizing these species.
• Lifespan Extension: Supplementation with $NaHS$ or $Na_2S_2$ extended the lifespan of WT yeast but had no effect on $\Delta hmt2$, proving the effect is strictly SQR-dependent.

B. Mammalian ($Sqrdl^{\Delta N/\Delta N}$) Findings:

• Phenotype: Mice lacking mitochondrial SQR exhibited emaciation and premature death after weaning.
• Metabolomics: $Sqrdl^{\Delta N/\Delta N}$ iMEFs and mitochondria showed dramatic accumulation of hydropersulfides (CysSSH, GSSH, HSSH) and $H_2S$.
• Bioenergetics:
  • Mitochondrial membrane potential was significantly reduced in mutant cells.
  • While basal OCR was similar to WT, the ability to generate membrane potential via sulfide/persulfide oxidation was lost.
  • Inhibitor studies showed that SQR-mediated potential generation is independent of Complex I and II but dependent on Complex III.
• Substrate Validation:
  • Isolated mitochondria from WT mice generated membrane potential upon $NaHS$ or $Na_2S_2$ addition; this was absent in mutant mitochondria.
  • Crucially, the membrane potential generation persisted even in the presence of an $H_2S$ scavenger (SS19), indicating that free $H_2S$ is not the primary bioenergetic substrate. Instead, downstream supersulfides (hydropersulfides/polysulfides) are the functional substrates.
  • Oxidized glutathione trisulfide (GSSSG) was shown to generate GSSH, which serves as a substrate for SQR.

5. Significance

• Redefining Mitochondrial Energy Metabolism: The study challenges the dogma that mitochondrial energy production relies solely on NADH/FADH2 from the Krebs cycle. It proposes a "sulfur respiration" cycle where cysteine-derived supersulfides act as electron donors to CoQ, bypassing Complex I.
• Therapeutic Implications for Mitochondrial Diseases: Since this pathway bypasses Complex I, the authors hypothesize that activating sulfur respiration (e.g., via cystine supplementation or chemical supersulfide boosters) could rescue energy deficits in patients with Complex I deficiencies (e.g., Leigh syndrome).
• Longevity Mechanism: The study provides a mechanistic link between sulfide/hydropersulfide supplementation and longevity, showing that these effects are strictly dependent on functional SQR to convert sulfur species into bioenergetic reducing equivalents.
• Evolutionary Insight: It suggests that eukaryotes retain an evolutionary legacy of sulfide-based respiration, allowing them to adapt to varying metabolic states and potentially extend lifespan.

In conclusion, the paper identifies mitochondrial SQR as a critical regulator that converts toxic sulfur species into bioenergetic fuel, driving membrane potential and ATP production, and is essential for organismal longevity across eukaryotes.