In eukaryotes, hydrogen sulphide acts as a signalling molecule and cytoprotectant. Hydrogen sulphide is known to be produced from L-cysteine by cystathionine b-synthase, cystathionine g-lyase and 3-mercaptopyruvate sulfurtransferase coupled with cysteine aminotransferase. Here we report an additional biosynthetic pathway for the production of hydrogen sulphide from D-cysteine involving 3-mercaptopyruvate sulfurtransferase and D-amino acid oxidase. Unlike the L-cysteine pathway, this D-cysteine-dependent pathway operates predominantly in the cerebellum and the kidney. Our study reveals that administration of D-cysteine protects primary cultures of cerebellar neurons from oxidative stress induced by hydrogen peroxide and attenuates ischaemia-reperfusion injury in the kidney more than L-cysteine. This study presents a novel pathway of hydrogen sulphide production and provides a new therapeutic approach to deliver hydrogen sulphide to specific tissues.

Hydrogen sulfide (H(2)S) has been recognized as a smooth muscle relaxant. Cystathionine gamma-lyase, which is localized to smooth muscle, is thought to be the major H(2)S-producing enzyme in the thoracic aorta. Here we show that 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) are localized to vascular endothelium in the thoracic aorta and produce H(2)S. Both 3MST and CAT were localized to endothelium. Lysates of vascular endothelial cells produced H(2)S from cysteine and alpha-ketoglutarate. The present study provides a new insight into the production and release of H(2)S as a smooth muscle relaxant from vascular endothelium.

H2S (hydrogen sulfide) has recently been recognized as a signalling molecule as well as a cytoprotectant. We recently demonstrated that 3MST (3-mercaptopyruvate sulfurtransferase) produces H2S from 3MP (3-mercaptopyruvate). Although a reducing substance is required for an intermediate persulfide at the active site of 3MST to release H2S, the substance has not been identified. In the present study we show that Trx (thioredoxin) and DHLA (dihydrolipoic acid) associate with 3MST to release H2S. Other reducing substances, such as NADPH, NADH, GSH, cysteine and CoA, did not have any effect on the reaction. We also show that 3MST produces H2S from thiosulfate. The present study provides a new insight into a mechanism for the production of H2S by 3MST.

Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles. We recently found H2Sn in the brain. H2Sn induced some responses previously attributed to H2S but with much greater potency than H2S. However, the number of sulfur atoms in H2Sn and its producing enzyme were unknown. Here, we detected H2S3 and H2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain. High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H2S3 and H2S were produced from 3 MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not. H2S3 was localized in the cytosol of cells. H2S3 was also produced from H2S by 3MST and rhodanese. H2S2 was identified as a minor H2Sn, and 3 MP did not affect the H2S5 level. The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/ PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-Å resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

In our previous study, we found that mercaptopyruvate sulfurtransferase (MST) was evolutionarily related to mitochondrial rhodanese. To elucidate the difference between MST and rhodanese, the tissue, cellular, and subcellular distribution of rat MST was determined biochemically and immunohistochemically by using anti-MST antibody raised in rabbit. In an immunohistochemical study, tetramethyl rhodamine isothiocyanate-conjugated phalloidin against F-actin and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin as a secondary antibody to the anti-MST antibody were used for double fluorescent staining. They were detected by confocal laser fluorescence microscopy. In the immunoelectron microscopic study of hepatocyte and renal tubular epithelium, a postembedding immunogold method was used. Biochemical studies including western blot analyses of various tissues and subcellular fractions of the liver were also performed. MST was widely distributed in rat tissues but the cellular distribution was found to be different in each tissue. MST was predominantly localized in proximal tubular epithelium in the kidney, pericentral hepatocytes in the liver, cardiac cells in the heart, and neuroglial cells in the brain. This immunocytochemical study also found that MST was localized in both mitochondria and cytoplasm.

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A complete amino acid structure of rat liver mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) was determined by sequence analysis of cDNA and purified enzyme. The enzyme consists of 296 amino acid residues with a calculated molecular mass of 32,808 Da. Sequence identity in cDNA and the deduced amino acid sequence are 65 and 60% respectively, between rat MST and rhodanese. By their entire sequence similarity MST and rhodanese are confirmed to be evolutionarily related enzymes (Nagahara, N., Okazaki, T., and Nishino, T. (1995) J. Biol. Chem. 270, 16230 -16235). The conversion of MST to rhodanese was attempted, and the role of amino acid residues was studied by site-directed mutagenesis with the isolated cDNA of rat liver MST. In the reaction the outer sulfur atom of thiosulfate is transferred to the Cys residue of the enzyme molecule to form a persulfide intermediate, which is subsequently attacked by cyanide anion to give thiocyanide. However, the enzyme has rather wide substrate specificity. Certain other sulfur compounds such as thiosulfonate (2) or persulfides (3, 4) may substitute for thiosulfate; and sulfite (5), sulfinates (5), or various thiol compounds (6) may substitute for cyanide. This enzyme was reported to be widely distributed in prokaryote and eukaryote mitochondria (7-9). Although the physiological role of this enzyme is not well understood, the enzyme is well characterized. The enzyme was first isolated from bovine liver (10) and subsequently from rat liver (11). Primary structures of the enzyme from various sources were determined from protein or deduced from cDNA, e.g. bovine liver (12) and adrenal (13), chicken liver (14), human liver (15), rat liver (16), hamster ovary (17), and mouse liver (18). Further, recombinant bovine liver (13) and adrenal (19), rat liver (20), hamster ovary (17), and mouse liver (18) rhodanese were overexpressed in Escherichia coli and characterized. The enzyme from bovine liver was crystallized (10, 21), and the three-dimensional structure was determined. (22)(23)(24). On the other hand, mercaptopyruvate sulfurtransferase (MST, 1 EC 2.8.1.2) catalyzes the following reaction.The enzyme was discovered in rat liver quite a long time ago (25-28), but compared with rhodanese the enzyme was not well characterized. Although the enzyme responsible for this reaction was at first assumed to be rhodanese, subsequent investigation with crystallized rhodanese showed that rhodanese did not catalyze this reaction and that another enzyme, mercaptopyruvate sulfurtransferase, was responsible for the reaction (28). Similar to rhodanese, compounds other than cyanide may function as sulfur acceptors in the reaction catalyzed by this enzyme (29 -32), and its physiological role is a matter of discussion (33, 34). This enzyme was reported to be distributed in both prokaryotes and eukaryotes and to be located in the cytosol of eukaryotic cells (26,27, 34,35), but the existence of this enzyme in mitochondria is also reported (36). Recently rat liver MST was purified to homogeneity, and th...