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J. Biol. Chem., Vol. 283, Issue 15, 9642-9650, April 11, 2008

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

Silke Wollers, Torsten Heidenreich, Maryam Zarepour, Dieter Zachmann, Claudia Kraft, Yunde Zhao^¶, Ralf R. Mendel¹, and Florian Bittner

From the Department of Plant Biology and Department of Environmental Geology, Technical University of Braunschweig, 38023 Braunschweig, Germany and ^¶Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, California 92093-0116

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 NH₂-terminal NifS-like cysteine desulfurase domain and a C-terminal domain of yet undescribed function. The NH₂-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.

Received for publication, October 15, 2007 , and in revised form, December 18, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant Me 1266/16-3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed: Dept. of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38023 Braunschweig, Germany. Tel.: 49-531-391-5870; Fax: 49-531-391-8128; E-mail: r.mendel{at}tu-bs.de.

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