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J. Biol. Chem., Vol. 282, Issue 48, 34921-34928, November 30, 2007

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Observations on the Calcium Dependence and Reversibility of Cobalamin Transport across the Outer Membrane of Escherichia coli^*

Nathalie Cadieux¹, Nazir Barekzi, and Clive Bradbeer²

From the Departments of Microbiology and Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908-0733

The calcium dependence of cobalamin (Cbl) binding to the BtuB protein of Escherichia coli and the reversibility of its function in the transport of Cbl across the outer membrane have been examined. The results show that the two calcium-binding sites in BtuB that were identified previously by others are responsible for the calcium dependence of high affinity Cbl binding. The affinity of the pure BtuB protein for Cbl was 1000-fold higher in the presence of saturating levels of calcium than in its absence. The affinities of BtuB for both Cbl and calcium were decreased by insertion of alanine residues at position 51 of the mature protein and were increased by several mutations and deletions in the TonB box. Experiments on the uptake of Cbl into the periplasmic space showed that this process is reversible and that the exit of Cbl back into the medium does not require the protonmotive force. Our interpretation of these results is that the role of the TonB-ExbB-ExbD complex, potentiated by the protonmotive force, is to reduce the affinity of the Cbl-binding site, thus increasing the rate of Cbl release into the periplasmic space. The evidence also indicates that access of the Cbl-binding site of BtuB to the periplasmic space does not require removal of the hatch domain from the barrel.

Received for publication, September 5, 2007 , and in revised form, September 28, 2007.

^* This work was supported by National Institutes of Health Grant GM019078 (to R. J. Kadner). 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 Present address: Center for Biologics Evaluation and Research, FDA, Bethesda, MD 20892.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, University of Virginia, P. O. Box 800733, Charlottesville, VA 22908-0733. Tel.: 434-293-2640; Fax: 434-924-5069; E-mail: cb7f{at}virginia.edu.

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