The Ataxia telangiectasia-mutated and Rad3-related protein kinase regulates cellular hydrogen sulfide concentrations

The ataxia telangiectasia-mutated and Rad3-related (ATR) serine/threonine kinase plays a central role in the repair of replication-associated DNA damage, the maintenance of S and G2/M-phase genomic stability, and the promotion of faithful mitotic chromosomal segregation. A number of stimuli activate ATR, including persistent single-stranded DNA at stalled replication folks, R loop formation, hypoxia, ultraviolet light, and oxidative stress, leading to ATR-mediated protein phosphorylation. Recently, hydrogen sulfide (H₂S), an endogenous gasotransmitter, has been found to regulate multiple cellular processes through complex redox reactions under similar cell stress environments. Three enzymes synthesize H₂S: cystathionine-β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Since H₂S can under some conditions cause DNA damage, we hypothesized that ATR activity may regulate cellular H₂S concentrations and H₂S-syntheszing enzymes. Here we show that human colorectal cancer cells carrying biallelic knock-in hypomorphic ATR mutations have lower cellular H₂S concentrations than do syngeneic ATR wild-type cells, and all three H₂S-synthesizing enzymes show lower protein expression in the ATR hypomorphic mutant cells. Additionally, ATR serine 428 phosphorylation is altered by H₂S donor and H₂S synthesis enzyme inhibition, while the oxidative-stress induced phosphorylation of the ATR-regulated protein CHK1 on serine 345 is increased by H₂S synthesis enzyme inhibition. Lastly, inhibition of H₂S production potentiated oxidative stress-induced double-stranded DNA breaks in the ATR hypomorphic mutant compared to ATR wild-type cells. Our findings demonstrate that the ATR kinase regulates and is regulated by H₂S.     

The effect of three α-keto acids on 3-mercaptopyruvate sulfurtransferase activity

3-Mercaptopyruvate sulfurtransferase catalyzes the transfer of sulfur from 3-mercaptopyruvate to several possible acceptor molecules, one of which is cyanide. Because the transsulfuration of cyanide is the primary in vivo mechanism of detoxification, 3-mercaptopyruvate sulfurtransferase may function in the enzymatic detoxification of cyanide in vivo. Three α-keto acids (α-ketobutyrate, α-ketoglutarate, and pyruvate) have previously been demonstrated to be cyanide antidotes in vivo, and it has been suggested that this is due to the nonenzymatic binding of cyanide by the α-keto acid. However, it has also been proposed that α-keto acids may increase the activity of enzymes involved in the transsulfuration of cyanide. Thus, the effect of these three α-keto acids on the enzyme 3-mercaptopyruvate sulfurtransferase was examined. All three α-keto acids inhibited 3-mercaptopyruvate sulfurtransferase in a concentration-dependent manner and were determined to be uncompetitive inhibitors of MST with respect to 3-mercaptopyruvate. The inhibitor constant K_i was estimated by two methods for each inhibitor and ranged from 4.3 to 6.3 mM. The I₅₀, which is the inhibitor concentration that produces 50% inhibition, was calculated for all three α-keto acids and ranged between 9.5 and 13.7 mM. These observations add further support to the hypothesis that the mechanisms of the α-keto acid antidotes is the nonenzymatic binding of cyanide, not stimulation of enzymes involved in the transsulfuration of cyanide to thiocyanate. © 1996 John Wiley & Sons, Inc.     

The cytosolic Arabidopsis thaliana cysteine desulfurase ABA3 delivers sulfur to the sulfurtransferase STR18

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain–containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain–containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD–Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3–STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.     

Functional sulfurtransferase is associated with mitochondrial complex I from Yarrowia lipolytica, but is not required for assembly of its iron-sulfur clusters

Here, we report that in the obligate aerobic yeast Yarrowia lipolytica, a protein exhibiting rhodanese (thiosulfate:cyanide sulfurtransferase) activity is associated with proton pumping NADH:ubiquinone oxidoreductase (complex I). Complex I is a key enzyme of the mitochondrial respiratory chain that contains eight iron-sulfur clusters. From a rhodanese deletion strain, we purified functional complex I that lacked the additional protein but was fully assembled and displayed no functional defects or changes in EPR signature. In contrast to previous suggestions, this indicated that the sulfurtransferase associated with Y. lipolytica complex I is not required for assembly of its iron-sulfur clusters.     

Enzymatic activation of sulfur for incorporation into biomolecules in prokaryotes

Sulfur is a functionally important element of living matter. Incorporation into biomolecules occurs by two basic strategies. Sulfide is added to an activated acceptor in the biosynthesis of cysteine, from which methionine, coenzyme A and a number of biologically important thiols can be constructed. By contrast, the biosyntheses of iron sulfur clusters, cofactors such as thiamin, molybdopterin, biotin and lipoic acid, and the thio modification of tRNA require an activated sulfur species termed persulfidic sulfur (R-S-SH) instead of sulfide. Persulfidic sulfur is produced enzymatically with the IscS protein, the SufS protein and rhodanese being the most prominent biocatalysts. This review gives an overview of sulfur incorporation into biomolecules in prokaryotes with a special emphasis on the properties and the enzymatic generation of persulfidic sulfur as well as its use in biosynthetic pathways.     

Chemical aspects of hydrogen sulfide measurements in physiological samples

Background

Owing to recent discoveries of many hydrogen sulfide-mediated physiological processes, sulfide biology is in the focus of scientific research. However, the promiscuous chemical properties of sulfide pose complications for biological studies, which led to accumulation of controversial observations in the literature.

Scope of review

We intend to provide an overview of fundamental thermodynamic and kinetic features of sulfide redox- and coordination-chemical reactions and protonation equilibria in relation to its biological functions. In light of these chemical properties we review the strengths and limitations of the most commonly used sulfide detection methods and recently developed fluorescent probes. We also give a personal perspective on blood and tissue sulfide measurements based on proposed biomolecule–sulfide interactions and point out important chemical aspects of handling sulfide reagent solutions.

Major conclusions

The diverse chemistries of sulfide detection methods resulted in orders of magnitude differences in measured physiological sulfide levels. Investigations that were aimed to dissect the underlying molecular reasons responsible for these controversies made the important recognition that there are large sulfide reserves in biological systems. These sulfide pools are tightly regulated in a dynamic manner and they are likely to play a major role in regulation of endogenous-sulfide-mediated biological functions and avoiding toxic side effects.

General significance

Working with sulfide is challenging, because it requires considerable amounts of chemical knowledge to adequately handle reagent sulfide solutions and interpret biological observations. Therefore, we propose that a rigorous chemical approach could aid the reconciliation of the increasing number of controversies in sulfide biology. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Regional and Subcellular Distribution of Cyanide Metabolizing Enzymes in the Central Nervous System

Activities of cyanide metabolizing enzymes were measured in various subcellular fractions and regions in the central nervous system. Brain rhodanese and liver beta-mercaptopyruvate sulfurtransferase showed a slight decrease in activity after death. The activity of beta-mercaptopyruvate sulfurtransferase was negligible in the rat brain, compared with that of rhodanese. A small amount of thiocyanate was produced from cyanide and beta-mercaptopyruvate in the human brain, probably due to contamination with red blood cells. Rhodanese activity was widely distributed in all the areas of nervous tissue examined. In the rat the olfactory bulb showed the highest rhodanese activity, and high activity was also observed served in the thalamus, septum, hippocampus, and dorsal part of the midbrain. Rhodanese activity was low in various parts of the cerebral cortex. The distribution pattern of rhodanese in post-mortem human brain was essentially similar to that in rat brain. The thalamus, amygdala, centrum semiovale, colliculus superior, and cerebellar cortex showed high rhodanese activity in the human brain. Rhodanese activity was detected in the spinal cord. Anterior horn showed the highest rhodanese activity in the cervical, thoracic, and lumbar cord. Most rhodanese activity in the rat brain was recovered in the mitochondrial fraction with the highest specific activity. Rhodanese activity was lower in spinal cords obtained from autopsied cases with amyotrophic lateral sclerosis than in those of control subjects. A significant decrease in rhodanese was observed in the posterior column of the cervical or thoracic cord, but the activity in the anterior horn did not differ significantly between the two groups.     

Crystal structure of YnjE from Escherichia coli,a sulfurtransferase with three rhodanese domains

Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple‐domain rhodanese‐like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well‐characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N‐terminal inactive rhodanese‐like domain. Phylogenetic analysis reveals that YnjE triple‐domain homologs can be found in a variety of other γ‐proteobacteria, in addition, some single‐, tandem‐, four and even six‐domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3‐mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C‐terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.     

Rhodanese (thiosulfate:cyanide sulfurtransferase) from frog Rana temporaria

The molecular mass of rhodanese from the mitochondrial fraction of frog Rana temporaria liver, equaling 8.7 kDa, was determined by high-performance size exclusion chromatography (HP-SEC). The considerable difference in molecular weight and the lack of common antigenic determinants between frog liver rhodanese and bovine rhodanese suggest the occurrence of different forms of this sulfurtransferase in the liver of these animals.