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J. Biol. Chem., Vol. 282, Issue 20, 15126-15136, May 18, 2007

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Characterization of the Outer Membrane Receptor ShuA from the Heme Uptake System of Shigella dysenteriae

SUBSTRATE SPECIFICITY AND IDENTIFICATION OF THE HEME PROTEIN LIGANDS^*

From the Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201

Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors.

Received for publication, December 4, 2006 , and in revised form, March 19, 2007.

^* This work was supported by National Institutes of Health Grant AI-48551. 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 To whom correspondence should be addressed: Dept. of Pharmaceutical Sciences, School of Pharmacy, 20 Penn St., University of Maryland, Baltimore, MD 21201. Tel.: 410-706-2537; Fax: 410-706-5017; E-mail: awilks{at}rx.umaryland.edu.

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