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J. Biol. Chem., Vol. 281, Issue 30, 20761-20771, July 28, 2006

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The Mechanism of Direct Heme Transfer from the Streptococcal Cell Surface Protein Shp to HtsA of the HtsABC Transporter^*

Tyler K. Nygaard, George C. Blouin, Mengyao Liu, Maki Fukumura, John S. Olson, Marian Fabian, David M. Dooley^¶, and Benfang Lei¹

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

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K_d of 120 ± 18 µM, and transfers its heme to apoHtsA with a rate constant of 28 ± 6s^–1 at 25 °C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 ± 0.002 s^–1. HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp·apoHtsA complex (K_d = 48 ± 7 µM) at a rate 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k_transfer = 43 ± 3s^–1 versus k_–hemin = 0.0003 ± 0.00006 s^–1). The rate constants for hemin binding to and dissociation from HtsA (k'_hemin 80 µM^–1 s^–1, k_–hemin = 0.0026 ± 0.0002 s^–1) are 50- and 10-fold greater than the corresponding rate constants for Shp (k_hemin 1.6 µM^–1 s^–1, k_–hemin = 0.0003 s^–1), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k_hemin 31,000 µM^–1) is roughly 5-fold greater than that of apoShp (k_hemin 5,300 µM^–1), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.

Received for publication, February 27, 2006 , and in revised form, April 27, 2006.

^* This work was supported by National Institutes of Health Grants K22 AI057347 (to B. L.), RO1 HL47020 (to J. S. O.), RO1 GM35649 (to J. S. O.), and RO1 GM55807 (to M. F.) National Center for Research Resources Grant P20 RR-020185 (to B. L.), Robert A. Welch Foundation Grant C-0612 (to J. S. O.), 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.

The on-line version of this article (available at http://www.jbc.org) contains Fig. S1.

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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