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J. Biol. Chem., Vol. 282, Issue 43, 31380-31388, October 26, 2007

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Bis-methionine Ligation to Heme Iron in the Streptococcal Cell Surface Protein Shp Facilitates Rapid Hemin Transfer to HtsA of the HtsABC Transporter^*

Yanchao Ran, Hui Zhu, Mengyao Liu, Marian Fabian, John S. Olson, Roman Aranda, IV^¶, George N. Phillips, Jr.^||, David M. Dooley^**, and Benfang Lei¹

From the Departments of Veterinary Molecular Biology and ^**Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59718, the Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005, and the Departments of ^¶Biomolecular Chemistry and ^||Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

The surface protein Shp of Streptococcus pyogenes rapidly transfers its hemin to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. The structural basis and molecular mechanism of this hemin transfer have been explored by mutagenesis and truncation of Shp. The heme-binding domain of Shp is in the amino-terminal region and is functionally active by itself, although inclusion of the COOH-terminal domain speeds up the process 10-fold. Single alanine replacements of the axial methionine 66 and 153 ligands (Shp^M66A and Shp^M153A) cause formation of pentacoordinate hemin-Met complexes. The association equilibrium constants for hemin binding to wild-type, M66A, and M153A Shp are 5,300, 22,000, and 38 µM^-1, respectively, showing that the Met¹⁵³–Fe bond is critical for high affinity binding and that Met⁶⁶ destabilizes hemin binding to facilitate its rapid transfer. Shp^M66A and Shp^M153A rapidly bind to hemin-free HtsA (apoHtsA), forming stable transfer intermediates. These intermediates appear to be Shp-hemin-HtsA complexes with one axial ligand from each protein and decay to the products with rate constants of 0.4–3 s^-1. Thus, the M66A and M153A replacements alter the kinetic mechanism and unexpectedly slow down hemin transfer by stabilizing the intermediates. These results, in combination with the structure of the Shp heme-binding domain, allow us to propose a "plug-in" mechanism in which side chains from apoHtsA are inserted into the axial positions of hemin in Shp to extract it from the surface protein and pull it into the transporter active site.

Received for publication, July 20, 2007 , and in revised form, August 6, 2007.

^* This work was supported by National Institutes of Health Grants AI057347 (to B. L.), GM27659 (to D. M. D.), HL47020 (to J. S. O.), GM35649 (to J. S. O.), GM55807 (to M. F.), and GM08349 (to R. A.); National Center for Research Resources Grant P20 RR-020185 (to B. L.); Robert A. Welch Foundation Grant C-0612 (to J. S. O.); United States Department of Agriculture Grant NRI/CGP 2006-01690 and Formula Funds; and the Montana State University Agricultural Experimental Station. 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 Veterinary Molecular Biology, Montana State University, P.O. Box 173610, Bozeman, MT 59717. Tel.: 406-994-6389; Fax: 406-994-4303; E-mail: blei{at}montana.edu.

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