Switching ON Hydrogen Sulfide: A Chemogenetic Toolkit for Spatially Resolved H2S Manipulation

Switching ON Hydrogen Sulfide: A Chemogenetic Toolkit for Spatially Resolved H2S Manipulation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Switching ON Hydrogen Sulfide: A Chemogenetic Toolkit for Spatially Resolved H 2 S Manipulation Asal Ghaffari Zaki , Seyed Mohammad Miri , Emre Vatandaşlar , Sven Vilain , Esra Nur Yiğit , Mehmet Şerif Aydın , Muhammed İkbal Alp , Emrah Eroglu doi: https://doi.org/10.1101/2025.05.12.653628 Asal Ghaffari Zaki 1 Research Institute for Health Sciences and Technologies (SABITA), İstanbul Medipol University , İstanbul 34810, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site Seyed Mohammad Miri 1 Research Institute for Health Sciences and Technologies (SABITA), İstanbul Medipol University , İstanbul 34810, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site Emre Vatandaşlar 1 Research Institute for Health Sciences and Technologies (SABITA), İstanbul Medipol University , İstanbul 34810, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sven Vilain 1 Research Institute for Health Sciences and Technologies (SABITA), İstanbul Medipol University , İstanbul 34810, Türkiye 6 Department of Medicinal Biology, International School of Medicine, İstanbul Medipol University , İstanbul, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site Esra Nur Yiğit 1 Research Institute for Health Sciences and Technologies (SABITA), İstanbul Medipol University , İstanbul 34810, Türkiye 3 Department of Physiology, International School of Medicine, İstanbul Medipol University , İstanbul 34810, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mehmet Şerif Aydın 1 Research Institute for Health Sciences and Technologies (SABITA), İstanbul Medipol University , İstanbul 34810, Türkiye 4 Department of Histology, School of Medicine, İstanbul Medipol University , İstanbul 34810, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site Muhammed İkbal Alp 1 Research Institute for Health Sciences and Technologies (SABITA), İstanbul Medipol University , İstanbul 34810, Türkiye 5 Department of Physiology, School of Medicine, İstanbul Medipol University , İstanbul 34810, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site Emrah Eroglu 1 Research Institute for Health Sciences and Technologies (SABITA), İstanbul Medipol University , İstanbul 34810, Türkiye 2 Molecular Biology, Genetics, and Bioengineering Program, Sabanci University , İstanbul 34956, Türkiye Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: emrah.eroglu{at}medipol.edu.tr Abstract Full Text Info/History Metrics Preview PDF Abstract Hydrogen sulfide (H 2 S) is emerging as a multifaceted signalling molecule that shapes energy metabolism, vascular tone, cancer biology and neurodegeneration. Although fluorescent and genetically encoded sensors now allow real-time H 2 S imaging, tools for selective control of intracellular H 2 S remain scarce. Here we benchmark two substrate-based chemogenetic enzymes—the yeast D-amino-acid oxidase (mDAAO) and the Salmonella typhimurium D-cysteine desulfhydrase (stDCyD)—for controlled H 2 S production. Both enzymes catalyse the conversion of D-cysteine to H 2 S, yet only mDAAO concomitantly generates hydrogen peroxide, introducing an unwanted oxidative signal. In contrast, stDCyD produces exclusively H 2 S, thereby offering a clean and efficient means to elevate intracellular H 2 S in vitro . Our data position stDCyD as the superior chemogenetic actuator for dissecting H 2 S biology without confounding redox artefacts. Introduction Hydrogen sulfide (H 2 S) has emerged as a critical gasotransmitter alongside nitric oxide (NO) and carbon monoxide (CO), with growing recognition of its diverse roles in physiological and pathological processes 1 , 2 . Originally regarded as merely a toxic gas, H 2 S is now known to be endogenously produced in mammalian cells by enzymes such as cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MPST) 3 , regulating key biological functions ranging from vascular tone 4 to mitochondrial bioenergetics 3 , 5 and neuromodulation 6 . Dysregulation of H 2 S homeostasis has been implicated in a range of disorders, including cardiovascular disease 7 , cancer 8 , and neurodegenerative conditions such as Alzheimer’s disease 9 . Despite these advances, our ability to study H 2 S dynamics at the cellular level remains significantly limited. Traditional approaches to modulate intracellular H 2 S levels largely rely on genetic manipulations—such as the overexpression or knockout of H 2 S-producing enzymes 10 — or pharmacological interventions, including the administration of sulfide-releasing salts like sodium hydrosulfide (NaHS) or slow-releasing H 2 S donors 11 . However, these methods suffer from major drawbacks, including poor spatiotemporal resolution, lack of reversibility, and off-target effects that complicate the interpretation of results. Particularly in single-cell studies or in complex tissue environments, the inability to achieve precise, real-time control of H 2 S concentrations limits the scope of mechanistic investigations. In recent years we have witnessed the development of chemical probes and genetically encoded sensors for real-time detection of H 2 S 12 – 15 . These advances have provided valuable insights into the spatial and temporal dynamics of H 2 S signaling. However, detection alone is insufficient for functional studies; tools are needed that not only report on H 2 S levels but also enable active manipulation with high precision. A novel chemogenetic approach for intracellular H 2 S manipulation would overcome the limitations of exogenous donor administration, providing unprecedented spatiotemporal control and the possibility to dissect causality in H 2 S signaling pathways. In this study, we introduce and characterize two enzymes with potential for use as chemogenetic tools to manipulate intracellular H 2 S levels. We investigated a modified D-amino acid oxidase (mDAAO) and Salmonella typhimurium derived D-cysteine desulfhydrase (stDCyD) that, in the presence of D-cysteine, can generate H 2 S 16 , 17 . Beyond technical innovation, the ability to precisely manipulate H 2 S levels will have broad implications for understanding H 2 S signaling in both health and disease. In the vascular system, H 2 S is a known vasodilator modulating endothelial nitric oxide synthase (eNOS) 18 , 19 . In cancer, altered H 2 S metabolism contributes to tumor progression, angiogenesis, and chemoresistance 20 . In the nervous system, H 2 S acts as a neuromodulator influencing synaptic transmission and neuroprotection 6 , but its dysregulation is associated with neurodegenerative diseases 21 , 22 . Thus, the development of a chemogenetic system for H 2 S manipulation is poised to fill a critical gap in the field, enabling novel experimental paradigms that were previously inaccessible. By introducing a robust, targeted, and highly specific strategy to control intracellular H 2 S concentrations, we provide a powerful tool to unravel the complex biology of this important gaseous signaling molecule. Results Figure 1 illustrates the distinct pathways by which the mDAAO and stDCyD enzymes mediate H 2 S production within cells. stDCyD, a pyridoxal phosphate (PLP)-dependent enzyme, catalyzes the direct conversion of D-cysteine into H 2 S, with pyruvate generated as a byproduct 1 7 . In contrast, mDAAO first oxidizes D-cysteine to its corresponding α-keto acid, namely mercaptopyruvate. Subsequently, mercaptopyruvate can be further metabolized to H 2 S by the endogenous enzyme mercaptopyruvate sulfurtransferase (MPST) 16 , 23 . By integrating these enzymatic H 2 S generation strategies with the genetically encoded biosensor hsGFP 13 , we establish a system that enables both dynamic production and real-time visualization of intracellular H 2 S ( Figure 1 ). Download figure Open in new tab Figure 1. Schematic representation of two chemogenetic strategies for H 2 S generation. The bacterial enzyme stDCyD directly metabolizes D-cysteine into H 2 S, which can be detected using the genetically encoded H 2 S sensor, hsGFP. Alternatively, the modified D-amino acid oxidase (mDAAO) first converts D-cysteine into 3-mercaptopyruvate (3-MP) while generating H 2 O 2 as a byproduct. The endogenous enzyme mercaptopyruvate sulfur transferase (3-MPST) subsequently converts 3-MP into H 2 S, which is also detectable by the hsGFP biosensor. Given that both the endogenous enzyme MPST and stDCyD generate pyruvate as a byproduct during H 2 S production 17 , 24 , we sought to assess pyruvate formation as an indirect measure of enzymatic activity and efficiency. To this end, we employed the genetically encoded pyruvate biosensor Pyronic 25 for real-time monitoring of intracellular pyruvate levels ( Figure 2A ). Upon administration of 5 mM D-cysteine, real-time imaging revealed no significant changes in pyruvate levels in either wild-type (WT) cells or cells expressing mCherry-mDAAO ( Figure 2B,C ). In contrast, cells expressing DsRed-stDCyD exhibited an increase in pyruvate generation ( Figure 2C , middle panel ), indicating a higher catalytic efficiency of stDCyD in metabolizing D-cysteine. Since the production of H 2 S and pyruvate by stDCyD occurs in equimolar amounts, these findings further support the suitability of stDCyD as an effective tool for controlled H 2 S generation. Download figure Open in new tab Figure 2. Pyruvate as a byproduct of enzymatic activity of stDCyD. A) Wide-field images of HEK 293T cells co-expressing DsRed-stDCyD (left panel), and Pyronic biosensor (middle and the right panels), scale bar represents 50 μm. B) Real-time traces of Pyruvate generation in HEK 293T cells co-expressing DsRed-stDCyD and Pyronic (red curve, 3/29) versus cells co-expressing mCherry-mDAAO and Pyronic (grey curve, 3/34), and cells only expressing Pyronic (green curve, 3/41) in presence of 5 mM D-Cysteine. At the end of the experiment cells were perfused with 5 mM pyruvate. C) One-way ANOVA analysis of FRET ratio in response to 5 mM D-cysteine and 5 mM pyruvate relative to the basal ratio. Previous work, including our own, has established that mDAAO generates hydrogen peroxide (H 2 O 2 ) during the oxidation of D-amino acids to the2ir corresponding α-keto acids 26 , 27 . To directly compare H 2 O 2 production between mDAAO and stDCyD, we co-transfected HEK 293 cells with plasmids encoding either mCherry-mDAAO or DsRed-stDCyD, together with the ratiometric H 2 O 2 biosensor HyPer7.2 28 . Upon acute administration of 5 mM D-cysteine, real-time imaging revealed a robust increase in intracellular H 2 O 2 levels in cells expressing mCherry-mDAAO, consistent with the enzyme’s oxidative activity. In contrast, cells expressing DsRed-stDCyD exhibited no detectable change in H 2 O 2 levels under the same conditions. These findings demonstrate that, unlike mDAAO, stDCyD enables selective H 2 S generation without concomitant oxidative stress, positioning it as a superior chemogenetic tool for controlled intracellular H 2 S modulation. Based on these findings, we selected stDCyD for subsequent experiments. As the next step, we aimed to directly visualize H 2 S generation resulting from stDCyD activity using the genetically encoded H 2 S biosensor, hsGFP 13 . WT HEK 293T cells and cells expressing stDCyD were transiently transfected with the hsGFP sensor. Upon acute administration of 10 mM D-cysteine, an increase in H 2 S levels was observed in both WT and stDCyD-expressing cells. However, the maximum response was significantly greater in cells expressing stDCyD compared to WT cells. These results provide direct evidence that stDCyD efficiently metabolizes D-cysteine to generate H 2 S and validate its utility as a robust tool for intracellular H 2 S production. Discussion Similar to H 2 O 2 , H 2 S was historically regarded as a toxic molecule 29 . Only in recent years critical role of H 2 S in cellular physiology been recognized 2 . This shift in understanding has catalyzed significant efforts to develop both chemical and genetically encoded probes for the real-time visualization of this gasotransmitter in vitro and in vivo . Current literature indicates that H 2 S exerts both beneficial and harmful effects depending on its concentration and the duration of exposure. At low concentrations, H 2 S supports various cellular processes, including signaling, energy metabolism, and protection against oxidative stress. However, at higher concentrations, it can exert cytotoxic effects and contribute to oxidative damage 3 . Thus, the ability to dynamically and precisely manipulate intracellular H 2 S levels represents an important unmet need in the field. Existing approaches to modulate H 2 S levels include the genetic manipulation of endogenous enzymes—such as CBS, CSE, and MPST—the application of H 2 S-releasing chemical cages, and the administration of sulfide salts (e.g., sodium hydrosulfide, NaHS). However, these strategies have notable limitations, including irreversibility, poor suitability for in vivo 30 use, off-target effects 20 , and a lack of spatiotemporal precision. Therefore, in this study, we have established a more efficient, reversible, and non-invasive method for the controlled generation of intracellular H 2 S. We evaluated two candidate enzymes for this purpose: mDAAO and stDCyD. In our previous work, we demonstrated that mDAAO can catalyze H 2 S production in the presence of D-cysteine 16 . Independently, the literature has also reported that stDCyD can generate H 2 S using D-cysteine as a substrate 17 . However, the mechanisms by which these enzymes produce H 2 S are distinct ( Figure 1 ). stDCyD directly converts D-cysteine into H 2 S and pyruvate, utilizing PLP as a cofactor. In contrast, mDAAO first oxidizes D-cysteine into its corresponding α-keto acid, mercaptopyruvate, which is subsequently metabolized to H 2 S by endogenous MPST. Both MPST and stDCyD produce pyruvate as a byproduct. Therefore, to indirectly compare the efficiency of these two enzymatic pathways (as well as the endogenous pathway), we used the genetically encoded pyruvate biosensor Pyronic to monitor pyruvate production in real time ( Figure 2 ). Our results showed that significant pyruvate generation occurred only in cells expressing stDCyD, but not in WT cells or those expressing mDAAO. These findings suggest that stDCyD exhibits superior catalytic efficiency for D-cysteine metabolism under the tested conditions. Further characterization of the enzymatic activities focused on H 2 O 2 generation. It is well established that mDAAO, while catalyzing the oxidation of D-amino acids, produces H 2 O 2 as a byproduct. Using the ratiometric H 2 O 2 sensor HyPer7.2, we observed a robust increase in intracellular H 2 O 2 following acute administration of D-cysteine in cells expressing mDAAO. In contrast, cells expressing stDCyD showed no detectable H 2 O 2 production under the same conditions ( Figure 3 ). This distinction further highlighted the advantage of stDCyD for specific H 2 S manipulation without the confounding effects of oxidative stress induced by H 2 O 2 . Based on these observations, we selected stDCyD as the preferred chemogenetic tool for subsequent experiments. Download figure Open in new tab Figure 3. mDAAO, but not stDCyD, generates H 2 O 2 in the presence of D-cysteine. A) Wide-field images of HEK 293T cells co-expressing DsRed-stDCyD (left panel) and the HyPer7.2 biosensor (middle and right panels). Scale bar: 50 μm. B) Real-time traces of H 2 O 2 generation in HEK 293T cells co-expressing DsRed-stDCyD and HyPer7.2 (red curve, 3/36) versus cells co-expressing mCherry-mDAAO and HyPer7.2 (gray curve, 3/40) upon addition of 5 mM D-cysteine. At the end of the experiment, cells were perfused with 50 μM exogenous H 2 O 2 . C, D) One-way ANOVA analysis of HyPer7.2 ratio changes in response to 5 mM D-cysteine and 50 μM H 2 O 2 relative to the basal ratio. To directly visualize H 2 S generation by stDCyD, we employed the genetically encoded H 2 S biosensor, hsGFP. Acute administration of 10 mM D-cysteine resulted in a significant increase in the hsGFP signal in cells expressing stDCyD, confirming active H 2 S production. Interestingly, a modest H 2 S signal was also detected in WT cells upon D-cysteine treatment ( Figure 4 ). Two plausible explanations for this observation exist based on prior reports. First, endogenous D-amino acid oxidase may oxidize D-cysteine to mercaptopyruvate, which is then converted to H 2 S by MPST 23 . Second, D-cysteine may be racemized to L-cysteine by endogenous serine racemase 31 , after which it can be metabolized by canonical H 2 S-producing enzymes such as CBS and CSE. To determine the contribution of these pathways, we plan to generate knockout (KO) cell lines targeting key enzymes involved in these alternative routes. Download figure Open in new tab Figure 4. H 2 S generation in HEK 293T cells upon enzymatic activity of stDCyD. A) Panel shows representative real-time traces of produced H 2 S in response to 10 mM D-Cysteine in HEK 293T cells co-expressing untargeted hsGFP and stDcyD (red curve, 3/41) or only hsGFP (green curve, 3/54). B) The bar graph shows statistical analysis of the maximum response of hsGFP in response to 10 mM D-Cysteine. Conclusion Our study introduces stDCyD as a robust, reversible, and precise tool for the intracellular generation of H 2 S, overcoming several limitations associated with existing methods. This platform will enable new investigations into the complex and dynamic roles of H 2 S in cellular physiology and pathophysiology. Materials and Methods Cell Culture Characterization experiments were conducted using cultured human embryonic kidney (HEK 293 T) cells maintained in a high-glucose (4.5 g/L) complete medium supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 U/ml penicillin, incubated at 37°C with 5% CO 2 . Approximately 24 hours before transfection, cells were plated onto 30 mm No.1 glass coverslips (Glaswarenfabrik Karl Knecht Sondheim, Germany). Once the cells reached about 70– 80% confluency, they were co-transfected with the appropriate mammalian expression plasmids using PolyJet transfection reagent, following the manufacturer’s protocol. Imaging was carried out 24 hours post-transfection. H 2 S imaging using hsGFP biosensor For H 2 S detection, cells were seeded onto 30 mm glass coverslips approximately 24 hours prior to transfection. Upon reaching ∼70–80% confluency, cells were co-transfected with the pMAH-POLY and the hsGFP biosensor plasmids using PolyJet transfection reagent. Five hours post-transfection, the culture medium was replaced with L-methionine and L-cystine-free DMEM supplemented with 2 mM p -azidophenylalanine ( P AZF) to enable non-canonical amino acid incorporation. After 24 hours of incubation, residual P AZF was removed by exchanging the medium for standard DMEM lacking L-methionine and L-cystine. Imaging experiments were conducted 24 hours following the P AZF washout. Buffers and Solutions Unless otherwise stated all chemicals were purchased from NeoFroxx. To maintain cells outside of the cell culture incubator, a cell storage buffer containing 2 mM CaCl 2 , 5 mM KCl, 138 mM NaCl, 1 mM MgCl 2 , 10 mM HEPES, 0.44 mM KH 2 PO 4 , 2.6 mM NaHCO 3 , 0.34 mM NaH2PO4, 10 mM D-Glucose, 0.1% MEM Vitamins (Pan-Biotech, Aidenbach, Germany), 0.2% essential amino acids (Pan-Biotech, Aidenbach, Germany), 100 μg/mL Penicillin (Pan-Biotech, Aidenbach, Germany), and 100 U/mL Streptomycin (Pan-Biotech, Aidenbach, Germany) was used. The pH was adjusted to 7.42 using 1 M NaOH. For live-cell imaging experiments, a HEPES buffer solution was used which consisted 2 mM CaCl 2 , 5 mM KCl, 138 mM NaCl, 1 mM MgCl 2 , 10 mM HEPES, 10 mM D-Glucose, and pH was adjusted to 7.42 using 1 M NaOH. Widefield epifluorescence microscopy Widefield epifluorescence microscopy was conducted using the Axio Observer Z1.7 inverted system (Carl Zeiss AG, Oberkochen, Germany), featuring a high-resolution Plan-Apochromat 40×/1.4 numerical aperture oil immersion lens and a monochrome Axiocam 503 CCD camera. A custom-built, gravity-based perfusion apparatus was integrated to enable precise delivery and removal of experimental solutions. For visualization of HyPer7-expressing cells, excitation was alternated between 423/44 nm and 469/38 nm wavelengths via a motorized filter wheel fitted with FT455 and FT495 dichroic beamsplitters (HyPer Low and High channels, respectively). Emission signals were collected through a common bandpass filter (525/50 nm) to facilitate ratiometric imaging. Cells expressing mcherry-mDAAO or DsRed-stDyCD were illuminated with 555/30 nm light, and emitted fluorescence was filtered through an FT570 dichroic mirror and 605/70 nm bandpass filter. FRET-based pyruvate sensor imaging was performed using 430 nm excitation, with emissions monitored at 480 nm and 525 nm for CFP and FRET channels respectively. Detection of H 2 S levels with the hsGFP biosensor was achieved by exciting at 480 nm and capturing emission at 525 nm. Image acquisition and system control were executed using Zen Blue 3.1 Pro software (Carl Zeiss AG). Statistical Analysis Imaging data were processed using GraphPad Prism version 5 (GraphPad Software, San Diego, CA, USA). Each experiment was repeated a minimum of three times, with results reported as N/n—where N denotes the number of independent experiments and n the number of cells analyzed. Data are presented as mean ± SEM, unless specified otherwise. For comparisons involving more than two groups, statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test (comparing each group to the control). For comparisons between two groups, an unpaired Student’s t-test was applied. Funding EMBO Installation Grant (EMBO IG J4113) to E.E. Conflicts of Interest The authors declare no conflict of interest. Acknowledgments Figure 1 was generated with BioRender.com (Agreement Number: BT289BFFTZ) Funder Information Declared European Molecular Biology Organization, https://ror.org/04wfr2810 , J4113 References 1. ↵ Li , L. , Rose , P. & Moore , P. K. Hydrogen Sulfide and Cell Signaling . Annual Review of Pharmacology and Toxicology 51 , 169 – 187 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ Cirino , G. , Szabo , C. & Papapetropoulos , A. Physiological roles of hydrogen sulfide in mammalian cells, tissues, and organs . Physiological Reviews 103 , 31 – 276 ( 2023 ). OpenUrl CrossRef PubMed 3. ↵ Paul , B. D. , Snyder , S. H. & Kashfi , K. Effects of hydrogen sulfide on mitochondrial function and cellular bioenergetics . Redox Biology 38 , 101772 ( 2021 ). OpenUrl CrossRef PubMed 4. ↵ Eberhardt , M. et al. H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO–TRPA1–CGRP signalling pathway . Nat Commun 5 , 4381 ( 2014 ). OpenUrl CrossRef PubMed 5. ↵ Fu , M. et al. Hydrogen sulfide (H2S) metabolism in mitochondria and its regulatory role in energy production . Proceedings of the National Academy of Sciences 109 , 2943 – 2948 ( 2012 ). OpenUrl Abstract / FREE Full Text 6. ↵ Panthi , S. , Chung , H.-J. , Jung , J. & Jeong , N. Y. Physiological Importance of Hydrogen Sulfide: Emerging Potent Neuroprotector and Neuromodulator . Oxidative Medicine and Cellular Longevity 2016 , 9049782 ( 2016 ). OpenUrl CrossRef 7. ↵ Shen , Y. , Shen , Z. , Luo , S. , Guo , W. & Zhu , Y. Z. The Cardioprotective Effects of Hydrogen Sulfide in Heart Diseases: From Molecular Mechanisms to Therapeutic Potential . Oxidative Medicine and Cellular Longevity 2015 , 925167 ( 2015 ). OpenUrl 8. ↵ Oza , P. P. & Kashfi , K. The Triple Crown: NO, CO, and H2S in cancer cell biology . Pharmacology & Therapeutics 249 , 108502 ( 2023 ). OpenUrl CrossRef PubMed 9. ↵ Paul , B. D. & Pieper , A. A. Neuroprotective signaling by hydrogen sulfide and its dysregulation in Alzheimer’s disease . Current Opinion in Chemical Biology 82 , 102511 ( 2024 ). OpenUrl CrossRef PubMed 10. ↵ Shaposhnikov , M. et al. Overexpression of CBS and CSE genes affects lifespan, stress resistance and locomotor activity in Drosophila melanogaster . Aging 10 , 3260 – 3272 ( 2018 ). OpenUrl CrossRef PubMed 11. ↵ Powell , C. R. , Dillon , K. M. & Matson , J. B. A review of hydrogen sulfide (H2S) donors: Chemistry and potential therapeutic applications . Biochem Pharmacol 149 , 110 – 123 ( 2018 ). OpenUrl CrossRef PubMed 12. ↵ Guo , M.-Y. et al. A structural optimized fluorescent probe for monitoring hydrogen sulfide in cells and zebrafish . Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 309 , 123763 ( 2024 ). OpenUrl CrossRef PubMed 13. ↵ Chen , Z. & Ai , H. A Highly Responsive and Selective Fluorescent Probe for Imaging Physiological Hydrogen Sulfide . Biochemistry 53 , 5966 – 5974 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 14. Lin , V. S. , Chen , W. , Xian , M. & Chang , C. J. Chemical probes for molecular imaging and detection of hydrogen sulfide and reactive sulfur species in biological systems . Chem. Soc. Rev . 44 , 4596 – 4618 ( 2015 ). OpenUrl CrossRef PubMed 15. ↵ Youssef , S. , Zhang , S. & Ai , H. A Genetically Encoded, Ratiometric Fluorescent Biosensor for Hydrogen Sulfide . ACS Sens . 4 , 1626 – 1632 ( 2019 ). OpenUrl CrossRef PubMed 16. ↵ Erdogan , Y. C. et al. Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting . Free Radical Biology and Medicine 177 , 132 – 142 ( 2021 ). OpenUrl CrossRef PubMed 17. ↵ Bharath , S. R. , Bisht , S. , Harijan , R. K. , Savithri , H. S. & Murthy , M. R. N. Structural and Mutational Studies on Substrate Specificity and Catalysis of Salmonella typhimurium D-Cysteine Desulfhydrase . PLOS ONE 7 , e36267 ( 2012 ). OpenUrl CrossRef PubMed 18. ↵ Altaany , Z. , Ju , Y. , Yang , G. & Wang , R. The coordination of S-sulfhydration, S-nitrosylation, and phosphorylation of endothelial nitric oxide synthase by hydrogen sulfide . Science Signaling 7 , ra87 – ra87 ( 2014 ). OpenUrl Abstract / FREE Full Text 19. ↵ King , A. L. et al. Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent . Proceedings of the National Academy of Sciences 111 , 3182 – 3187 ( 2014 ). OpenUrl Abstract / FREE Full Text 20. ↵ Cao , X. et al. A Review of Hydrogen Sulfide Synthesis, Metabolism, and Measurement: Is Modulation of Hydrogen Sulfide a Novel Therapeutic for Cancer? Antioxidants & Redox Signaling 31 , 1 – 38 ( 2019 ). OpenUrl CrossRef PubMed 21. ↵ Paul , B. D. et al. Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington’s disease . Nature 509 , 96 – 100 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 22. ↵ Tripathi , S. J. , Chakraborty , S. , Miller , E. , Pieper , A. A. & Paul , B. D. Hydrogen sulfide signalling in neurodegenerative diseases . British Journal of Pharmacology n/a ,. 23. ↵ Shibuya , N. et al. A novel pathway for the production of hydrogen sulfide from D-cysteine in mammalian cells . Nat Commun 4 , 1366 ( 2013 ). OpenUrl CrossRef PubMed 24. ↵ Pedre , B. et al. 3-Mercaptopyruvate sulfur transferase is a protein persulfidase . Nat Chem Biol 19 , 507 – 517 ( 2023 ). OpenUrl CrossRef PubMed 25. ↵ Martín , A. S. et al. Imaging Mitochondrial Flux in Single Cells with a FRET Sensor for Pyruvate . PLOS ONE 9 , e85780 ( 2014 ). OpenUrl CrossRef PubMed 26. ↵ Maurer , W. et al. D-amino acid oxidase as a chemogenetic tool for spatiotemporally controlled hydrogen peroxide production: The oxygen connection . British Journal of Pharmacology n/a ,. 27. ↵ Yadav , S. et al. Sensory ataxia and cardiac hypertrophy caused by neurovascular oxidative stress in chemogenetic transgenic mouse lines . Nat Commun 14 , 3094 ( 2023 ). OpenUrl CrossRef PubMed 28. ↵ Pak , V. V. et al. Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function . Cell Metabolism 31 , 642 - 653 .e6 ( 2020 ). OpenUrl CrossRef PubMed 29. ↵ Szabo , C. A timeline of hydrogen sulfide (H2S) research: From environmental toxin to biological mediator . Biochemical Pharmacology 149 , 5 – 19 ( 2018 ). OpenUrl CrossRef PubMed 30. ↵ Ghasemi , A. , Jeddi , S. , Yousefzadeh , N. , Kashfi , K. & Norouzirad , R. Dissolving sodium hydrosulfide in drinking water is not a good source of hydrogen sulfide for animal studies . Sci Rep 13 , 21839 ( 2023 ). OpenUrl CrossRef PubMed 31. ↵ Semenza , E. R. et al. D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain . Proceedings of the National Academy of Sciences 118 , e2110610118 ( 2021 ). OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted May 15, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Switching ON Hydrogen Sulfide: A Chemogenetic Toolkit for Spatially Resolved H2S Manipulation Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Switching ON Hydrogen Sulfide: A Chemogenetic Toolkit for Spatially Resolved H 2 S Manipulation Asal Ghaffari Zaki , Seyed Mohammad Miri , Emre Vatandaşlar , Sven Vilain , Esra Nur Yiğit , Mehmet Şerif Aydın , Muhammed İkbal Alp , Emrah Eroglu bioRxiv 2025.05.12.653628; doi: https://doi.org/10.1101/2025.05.12.653628 Share This Article: Copy Citation Tools Switching ON Hydrogen Sulfide: A Chemogenetic Toolkit for Spatially Resolved H 2 S Manipulation Asal Ghaffari Zaki , Seyed Mohammad Miri , Emre Vatandaşlar , Sven Vilain , Esra Nur Yiğit , Mehmet Şerif Aydın , Muhammed İkbal Alp , Emrah Eroglu bioRxiv 2025.05.12.653628; doi: https://doi.org/10.1101/2025.05.12.653628 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biochemistry Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17635) Bioengineering (13859) Bioinformatics (41846) Biophysics (21401) Cancer Biology (18534) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24285) Genetics (15582) Genomics (22463) Immunology (17700) Microbiology (40298) Molecular Biology (17141) Neuroscience (88424) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4813) Physiology (7633) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)