Binding of Sulfurated Molybdenum Cofactor to the C-terminal Domain of ABA3 from Arabidopsis thaliana Provides Insight into the Mechanism of Molybdenum Cofactor Sulfuration

Abstract

The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana is needed for post-translational activation of aldehyde oxidase and xanthine dehydrogenase by transferring a sulfur atom to the desulfo-molybdenum cofactor of these enzymes. ABA3 is a two-domain protein consisting of an NH 2 -terminal NifS-like cysteine desulfurase domain and a C-terminal domain of yet undescribed function. The NH 2 -terminal domain of ABA3 decomposes L-cysteine to yield elemental sulfur, which subsequently is bound as persulfide to a conserved protein cysteinyl residue within this domain. In vivo, activation of aldehyde oxidase and xanthine dehydrogenase also depends on the function of the C-terminal domain, as can be concluded from the A. thaliana aba3/sir3-3 mutant. sir3-3 plants are strongly reduced in aldehyde oxidase and xanthine dehydrogenase activities due to a substitution of arginine 723 by a lysine within the C-terminal domain of the ABA3 protein. Here we present first evidence for the function of the C-terminal domain and show that molybdenum cofactor is bound to this domain with high affinity. Furthermore, cyanide-treated ABA3 C terminus was shown to release thiocyanate, indicating that the molybdenum cofactor bound to the C-terminal domain is present in the sulfurated form. Co-incubation of partially active aldehyde oxidase and xanthine dehydrogenase with ABA3 C terminus carrying sulfurated molybdenum cofactor resulted in stimulation of aldehyde oxidase and xanthine dehydrogenase activity. The data of this work suggest that the C-terminal domain of ABA3 might act as a scaffold protein where prebound desulfo-molybdenum cofactor is converted into sulfurated cofactor prior to activation of aldehyde oxidase and xanthine dehydrogenase.Molybdenum enzymes catalyze diverse redox reactions in the global carbon, nitrogen, and sulfur cycles (1). In all eukaryotic molybdenum enzymes, the molybdenum atom is coordinated by the dithiolene group of molybdopterin, thus forming the molybdenum cofactor (Moco) 2 (2). According to the coordination chemistry of the molybdenum ligand, eukaryotic molybdenum enzymes can be divided into two groups; Moco with two additional oxo-ligands and a protein-derived cysteinyl sulfur is bound by enzymes of the sulfite oxidase family, whereas enzymes of the xanthine oxidase family have one oxygen, one inorganic sulfur, and one hydroxyl group ligated to the pterin-chelated molybdenum of the active enzyme. Among the four different molybdenum enzymes known in higher plants, sulfite oxidase and nitrate reductase belong to the sulfite oxidase family, whereas aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) are members of the xanthine oxidase family (3). Although it is believed that all of these molybdenum enzymes basically incorporate the same type of Moco, only AO and XDH, but not enzymes of the sulfite oxidase family, require a final enzyme-dependent post-translational modification of the molybdenum center for activity (4). During this modification step, an oxo-ligand of the Moco in inactive AO and XDH...

Sulphur shuttling across a chaperone during molybdenum cofactor maturation

“…So far, the precise reaction mechanism by which the inorganic sulphur of the cysteine-persulphide intermediate is transferred to the molybdenum atom of a bound cofactor remains unknown. Such a situation does not only applies to FdhD, but also to members of the xanthine oxidase family from both eukaryotes and prokaryotes, where a dedicated protein is in charge of molybdenum cofactor sulphuration prior to its insertion into the target enzymes 16,20,21 . It was also reported that sulphur transfer by persulphide chemistry may occur through two chemically reasonable routes 22,23 .…”

“…1b). The absorption around 400 nm may be attributed to the ene-dithiolate-to-molybdenum charge transfer band 16 . To further characterize the purified EcFdhD protein, the molybdenum content and the stable oxidation product derived from the cofactor (the so-called formA-dephospho) were quantified by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC) detection, respectively (Supplementary Table 1).…”

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

“…15-17 and this work). Although the Moco sulfurase consists of an N-terminal pyridoxal phosphate-binding domain responsible for abstracting sulfur from L-cysteine and a C-terminal Moco-binding domain onto which the sulfur is transferred (11,12), both hmARC proteins are stand-alone proteins lacking the Moco sulfurase-typical N-terminal domain. Interestingly, all eukaryotic organisms, with the exception of certain specialized Moco-independent yeast species (38), appear to harbor the same set of MOSC family proteins, suggesting a highly conserved function for each of these proteins.…”

“…The molybdenum-sulfur bond is essential for activity and is inserted in a post-translational reaction by a specific enzyme, the so-called Moco sulfurase (8 -10). The N-terminal domain of this enzyme is responsible for the mobilization of sulfur from L-cysteine (11), whereas the C-terminal domain binds Moco and is presumed to function as a scaffold for sulfuration of the cofactor as required by the enzymes of the XO family (12).…”

The Molybdenum Cofactor

“…In a pyridoxal phosphate-dependent manner, the N-terminal domain of ABA3 decomposes L-cysteine to yield alanine and elemental sulfur (57), the latter being bound as a persulfide to a highly conserved cysteine residue of ABA3. The C-terminal domain of ABA3 shares a significant degree of similarity with the newly discovered mitochondrial amidoxime-reducing component proteins and was shown to bind sulfurated Moco, which receives the terminal sulfur via an intramolecular persulfide relay from the N-terminal domain (58,59). It is likely that subsequent to Moco sulfuration, ABA3 exchanges non-sulfurated for sulfurated Moco, thus activating its target molybdenum enzyme.…”