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J. Biol. Chem., Vol. 282, Issue 14, 10380-10386, April 6, 2007

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Flavin Binding to the High Affinity Riboflavin Transporter RibU^*

From the Department of Biochemistry, University of Groningen, Groningen Biomolecular Science and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands

The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B₂) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method (Cadene, M., and Chait, B. T. (2000) Anal. Chem. 72, 5655–5658) to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate-free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1:1 stoichiometry (K_d for riboflavin is 0.6 nM), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin-binding proteins of known structure.

Received for publication, September 6, 2006 , and in revised form, February 6, 2007.

^* This work was supported in part by a Vidi grant from the Netherlands Organization for Scientific Research (to D. J. S.) and by the Netherlands Proteomics Center. 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.

¹ Supported by the EU-P6 programme E-Mep.

² To whom correspondence should be addressed. Tel.: 31-50-3634187; Fax: 31-50-3634165; E-mail: d.j.slotboom{at}rug.nl.

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