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 ;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.

The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind The molybdenum cofactor (Moco)2 is a prosthetic group highly conserved in all kingdoms of life and consists of a tricyclic pterin, referred to as molybdopterin or metal-binding pterin (MPT) and a molybdenum (Mo) atom covalently bound to the dithiolate moiety of MPT (1). Moco is required for the activity of all Mo-dependent enzymes with the exception of nitrogenase (2). Molybdenum enzymes (Mo-enzymes) are essential for a broad variety of metabolic processes such as nitrate assimilation and phytohormone synthesis in plants (3) and sulfur detoxification and purine catabolism in mammals (4).Synthesis of Moco proceeds in a highly conserved multistep pathway, involving at least six proteins named Cnx in plants (3). Much is known about the final step of Moco biosynthesis where one Mo atom is ligated to the MPT dithiolate function, which is catalyzed by the two-domain protein Cnx1 (5, 6): the C-terminal Cnx1-G domain activates MPT by adenylation, which is handed over to the N-terminal Cnx1-E domain where it is converted to Moco by inserting Mo into MPT under simultaneous cleavage of the pyrophosphate bond.After completion of biosynthesis, Moco has to be allocated and inserted into the apoMo-enzymes. In prokaryotes, a complex of proteins synthesizing the last steps of Moco biosynthesis donates the mature cofactor to apoenzymes assisted by enzyme-specific chaperones (7). In eukaryotes, however, no Mo-enzyme-specific chaperone has been found. As free Moco is extremely sensitive to oxidation it is also assumed that Moco occurs permanently protein-bound in the cell. Therefore, a cellular Moco distribution system should meet two demands: (i) it should bind Moco subsequent to its synthesis, and (ii) it should maintain a directed flow of Moco from the Moco donor Cnx1-E to the Mo-dependent enzymes. This is important to ensure the fast and efficient incorporation of Moco into apoMo-enzymes. In the alga Chlamydomonas reinhardtii a Moco carrier protein (MCP) was identified that was found to bind Moco and protect it against oxidation (8 -10). Without any denaturing procedure, subsequent transfer of Moco from MCP to apo-nitrate reductase (NR) from Neurospora crassa mutant nit-1 was possible (10), thus indicating that MCP-bound Moco was readily transferable. These properties of Chlamydomonas MCP make it a promising candidate for being part of a cellular Moco delivery system. It is, however, unknown whether MCP is also able to donate Moco to Mo-enzymes other than NR.Here we present the cloning and characterization of Mocobinding proteins (MoBP)

The molybdenum cofactor (Moco) is the active compound at the catalytic site of molybdenum enzymes. Moco is synthesized by a conserved four-step pathway involving six proteins in Arabidopsis thaliana. Bimolecular fluorescence complementation was used to study the subcellular localization and interaction of those proteins catalysing Moco biosynthesis. In addition, the independent split-luciferase approach permitted quantification of the strength of these protein–protein interactions in vivo. Moco biosynthesis starts in mitochondria where two proteins undergo tight interaction. All subsequent steps were found to proceed in the cytosol. Here, the heterotetrameric enzyme molybdopterin synthase (catalysing step two of Moco biosynthesis) and the enzyme molybdenum insertase, which finalizes Moco formation, were found to undergo tight protein interaction as well. This cytosolic multimeric protein complex is dynamic as the small subunits of molybdopterin synthase are known to go on and off in order to become recharged with sulphur. These small subunits undergo a tighter protein contact within the enzyme molybdopterin synthase as compared with their interaction with the sulphurating enzyme. The forces of each of these protein contacts were quantified and provided interaction factors. To confirm the results, in vitro experiments using a technique combining cross-linking and label transfer were conducted. The data presented allowed the outline of the first draft of an interaction matrix for proteins within the pathway of Moco biosynthesis where product–substrate flow is facilitated through micro-compartmentalization in a cytosolic protein complex. The protected sequestering of fragile intermediates and formation of the final product are achieved through a series of direct protein interactions of variable strength.