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Persulfide dioxygenases (PDOs) are abundant in Bacteria and also crucial for H2S detoxification in mitochondria. One of the two pdo-genes of the acidophilic bacterium Acidithiobacillus caldus was expressed in Escherichia coli. The protein (AcPDO) had 0.77 ± 0.1 Fe/subunit and an average specific sulfite formation activity of 111.5 U/mg protein (Vmax) at 40°C and pH 7.5 with sulfur and GSH following Michaelis–Menten kinetics. KM for GSH and Kcat were 0.5 mM and 181 s−1, respectively. Glutathione persulfide (GSSH) as substrate gave a sigmoidal curve with a Vmax of 122.3 U/mg protein, a Kcat of 198 s−1 and a Hill coefficient of 2.3 ± 0.22 suggesting positive cooperativity. Gel permeation chromatography and non-denaturing gels showed mostly tetramers. The temperature optimum was 40–45°C, the melting point 63 ± 1.3°C in thermal unfolding experiments, whereas low activity was measurable up to 95°C. Site-directed mutagenesis showed that residues located in the predicted GSH/GSSH binding site and in the central hydrogen bond networks including the iron ligands are essential for activity. Among these, the R139A, D141A, and H171A variants were inactive concomitant to a decrease of their melting points by 3–8 K. Other variants were inactivated without significant melting point change. Two out of five cysteines are likewise essential, both of which lie presumably in close proximity at the surface of the protein (C87 and C224). MalPEG labeling experiments suggests that they form a disulfide bridge. The reducing agent Tris(2-carboxyethyl)phosphine was inhibitory besides N-ethylmaleimide and iodoacetamide suggesting an involvement of cysteines and the disulfide in catalysis and/or protein stabilization. Mass spectrometry revealed modification of C87, C137, and C224 by 305 mass units equivalent to GSH after incubation with GSSH and with GSH in case of the C87A and C224A variants. The results of this study suggest that disulfide formation between the two essential surface-exposed cysteines and Cys-S-glutathionylation serve as a protective mechanism against uncontrolled thiol oxidation and the associated loss of enzyme activity.

Ethylmalonic encephalopathy (EE) is a devastating infantile metabolic disorder affecting the brain, gastrointestinal tract, and peripheral vessels. High levels of ethylmalonic acid are detected in the body fluids, and cytochrome c oxidase activity is decreased in skeletal muscle. By use of a combination of homozygosity mapping, integration of physical and functional genomic data sets, and mutational screening, we identified GenBank D83198 as the gene responsible for EE. We also demonstrated that the D83198 protein product is targeted to mitochondria and internalized into the matrix after energy-dependent cleavage of a short leader peptide. The gene had previously been known as "HSCO" (for hepatoma subtracted clone one). However, given its role in EE, the name of the gene has been changed to "ETHE1." The severe consequences of its malfunctioning indicate an important role of the ETHE1 gene product in mitochondrial homeostasis and energy metabolism.

The sulfur dioxygenase ETHE1 oxidizes persulfides in the mitochondrial matrix and is involved in the degradation of L-cysteine and hydrogen sulfide. ETHE1 has an essential but as yet undefined function in early embryo development of Arabidopsis thaliana. In leaves, ETHE1 is strongly induced by extended darkness and participates in the use of amino acids as alternative respiratory substrates during carbohydrate starvation. Thus, we tested the effect of darkness on seed development in an ETHE1 deficient mutant in comparison to the wild type. Since ETHE1 knock-out is embryo lethal, the knock-down line ethe1-1 with about 1% residual sulfur dioxygenase activity was used for this study. We performed phenotypic analysis, metabolite profiling and comparative proteomics in order to investigate the general effect of extended darkness on seed metabolism and further define the specific function of the mitochondrial sulfur dioxygenase ETHE1 in seeds. Shading of the siliques had no morphological effect on embryogenesis in wild type plants. However, the developmental delay that was already visible in ethe1-1 seeds under control conditions was further enhanced in the darkness. Dark conditions strongly affected seed quality parameters of both wild type and mutant plants. The effect of ETHE1 knock-down on amino acid profiles was clearly different from that found in leaves indicating that in seeds persulfide oxidation interacts with alanine and glycine rather than branched-chain amino acid metabolism. Sulfur dioxygenase deficiency led to defects in endosperm development possibly due to alterations in the cellularization process. In addition, we provide evidence for a potential role of persulfide metabolism in abscisic acid (ABA) signal transduction in seeds. We conclude that the knock-down of ETHE1 causes metabolic re-arrangements in seeds that differ from those in leaves. Putative mechanisms that cause the aberrant endosperm and embryo development are discussed.

Hydrogen sulfide is a physiologically relevant signalling molecule. However, circulating levels of this highly biologically active substance have to be maintained within tightly controlled limits in order to avoid toxic side effects. In patients suffering from EE (ethylmalonic encephalopathy), a block in sulfide oxidation at the level of the SDO (sulfur dioxygenase) ETHE1 leads to severe dysfunctions in microcirculation and cellular energy metabolism. We used an Ethe1-deficient mouse model to investigate the effect of increased sulfide and persulfide concentrations on liver, kidney, muscle and brain proteomes. Major disturbances in post-translational protein modifications indicate that the mitochondrial sulfide oxidation pathway could have a crucial function during sulfide signalling most probably via the regulation of cysteine S-modifications. Our results confirm the involvement of sulfide in redox regulation and cytoskeleton dynamics. In addition, they suggest that sulfide signalling specifically regulates mitochondrial catabolism of FAs (fatty acids) and BCAAs (branched-chain amino acids). These findings are particularly relevant in the context of EE since they may explain major symptoms of the disease.