A novel role for Arabidopsis mitochondrial ABC transporter ATM3 in molybdenum cofactor biosynthesis.

The molybdenum cofactor (Moco) is a prosthetic group required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Its biosynthesis in eukaryotes can be divided into four steps, of which the last three are proposed to occur in the cytosol. Here, we report that the mitochondrial ABC transporter ATM3, previously implicated in the maturation of extramitochondrial iron-sulfur proteins, has a crucial role also in Moco biosynthesis. In ATM3 insertion mutants of Arabidopsis thaliana, the activities of nitrate reductase and sulfite oxidase were decreased to approximately 50%, whereas the activities of xanthine dehydrogenase and aldehyde oxidase, whose activities also depend on iron-sulfur clusters, were virtually undetectable. Moreover, atm3 mutants accumulated cyclic pyranopterin monophosphate, the first intermediate of Moco biosynthesis, but showed decreased amounts of Moco. Specific antibodies against the Moco biosynthesis proteins CNX2 and CNX3 showed that the first step of Moco biosynthesis is localized in the mitochondrial matrix. Together with the observation that cyclic pyranopterin monophosphate accumulated in purified mitochondria, particularly in atm3 mutants, our data suggest that mitochondria and the ABC transporter ATM3 have a novel role in the biosynthesis of Moco.

SoxAX cytochromes, a new type of heme copper protein involved in bacterial energy generation from sulfur compounds.

SoxAX cytochromes are essential for the function of the only confirmed pathway for bacterial thiosulfate oxidation, the so-called "Sox pathway," in which they catalyze the initial formation of a S-S bond between thiosulfate and the SoxYZ carrier protein. Our work using the Starkeya novella diheme SoxAX protein reveals for the first time that in addition to two active site heme groups, SoxAX contains a mononuclear Cu(II) center with a distorted tetragonal geometry and three to four nitrogen ligands, one of which is a histidine. The Cu(II) center enhanced SoxAX activity in a newly developed, glutathione-based assay system that mimics the natural reaction of SoxAX with SoxYZ. EPR spectroscopy confirmed that the SoxAX Cu(II) center is reduced by glutathione. At pH 7 a K(m) (app) of 0.19+/-0.028 mm and a k(cat) (app) of 5.7+/-0.25s(-1) were determined for glutathione. We propose that SoxAX cytochromes are a new type of heme-copper proteins, with SoxAX-mediated S-S bond formation involving both the copper and heme centers.